CN101437848A - Selective VPAC2 receptor peptide agonists - Google Patents

Abstract

The present invention encompasses peptides that selectively activate the VPAC2 receptor and are useful in the treatment of diabetes.

Description

Selective VPAC2 receptor peptide agonist

The present invention relates to selective VPAC2 receptor peptide agonists.

In particular, the present invention relates to selective VPAC2 receptor peptide agonists comprising a C-terminal extension comprising the amino acid sequence GGPSSGAPPPK (EC ₁₆ ).

Type 2 diabetes, or non-insulin-dependent diabetes mellitus (NIDDM), is the most common form of diabetes, affecting 90% of people with diabetes. Patients with NIDDM have impaired β-cell function, resulting in insufficient insulin production and/or reduced insulin sensitivity. If NIDDM is not controlled, too much glucose can build up in the blood, leading to hyperglycemia. Over time, increasingly serious complications can develop, including renal dysfunction, cardiovascular problems, vision loss, leg ulcers, neuropathy, and ischemia. Treatment of NIDDM includes improved diet, exercise, weight control, and the use of a variety of oral medications. By taking such oral medications, individuals with NIDDM can initially control their blood sugar levels. However, this class of drugs does not slow the progressive loss of β-cell function in NIDDM patients and, therefore, is not sufficient to control blood glucose levels in advanced stages of the disease. Furthermore, treatment with currently available drugs exposes NIDDM patients to potential side effects such as hypoglycemia, gastrointestinal problems, fluid retention, edema, and/or weight gain.

The pituitary adenylate cyclase activating peptide (PACAP) and vasoactive intestinal peptide (VIP) belong to the same peptide family as eg secretin and glucagon. PACAP and VIP act through three G protein-coupled receptors and act through cAMP-mediated and other Ca2 ⁺ -mediated signal transduction pathways. Such receptors are known as PACAP-preferred type 1 (PAC1) receptors (Isobe et al., Regul. Pept., 110:213-217 (2003); Ogi et al., Biochem. Biophys. Res. Commun., 196:1511-1521 (1993)) and two VIP-shared type 2 receptors (VPAC1 and VPAC2) (Sherwood et al., Endocr. Rev., 21:619-670 (2000); Hammar et al., Pharmacol Rev. , 50:265-270 (1998); Couvineau et al., J.Biol.Chem., 278:24759-24766 (2003); Sreedharan, et al., Biochem.Biophys.Res.Commun., 193:546-553 ( 1993); Lutz et al., FEBS Lett., 458:197-203 (1999); Adamou et al., Biochem. Biophys. Res. Commun., 209:385-392 (1995)). A series of PACAP homologs are disclosed in US 6,242,563 and WO 2000/05260.

PACAP has comparable activity to all three receptors, whereas VIP selectively activates both VPAC receptors (Tsutsumi et al., Diobetes, 51:1453-1460 (2002)). VIP (Eriksson et al., Peptides, 10: 481-484 (1989)) and PACAP (Filipsson et al., JCEM, 82: 3093-3098 (1997)) have been shown to not only stimulate human insulin secretion , and increase glucagon secretion and hepatic glucose excretion. Thus, PACAP or VIP stimulation generally does not result in a net improvement in glycemia. Activation of multiple receptors by PACAP or VIP also has broad physiological effects on the nervous, endocrine, cardiovascular, reproductive, muscular and immune systems (Gozes et al., Curr. Med. Chem., 6:1019-1034( 1999)). Furthermore, VIP-induced watery diarrhea in rats was shown to be mediated only by one of the VPAC receptors, VPAC1 (Ito et al., Peptides, 22:1139-1151 (2001); Tsutsumi et al., Diabetes, 51:1453 -1460(2002)). Additionally VPAC1 and PAC1 receptors are expressed on α-cells and hepatocytes and are therefore most likely involved in hepatic glucose excretion.

Exendin-4 has been found in the salivary secretions of Heloderma Suspectum (Eng et al., J. Biol. Chem., 267(11):7402-7405 (1992)). This is a 39 amino acid peptide with glucose-dependent insulinotropic activity. Specific polyethylene glycol-incorporated exendins and exendin agonists are described in WO2000/66629. Exendin derivatives having at least one amino acid linked to a lipophilic substituent are described in WO99/43708.

Recent studies have shown that peptides that selectively act on the VPAC2 receptor can stimulate insulin secretion from the pancreas without gastrointestinal side effects and without increasing glucagon release and hepatic glucose excretion (Tsutsumi et al., Diabetes, 51:1453-1460 (2002)). Peptides acting selectively at the VPAC2 receptor were initially identified by modified VIP and/or PACAP. (See, e.g., Xia et al., JPharmacol Exp Ther., 281:629-633 (1997); Tsutsumi et al., Diabetes, 51:1453-1460 (2002); WO 01/23420; WO 2004/06839.)

However, many of the currently reported VPAC2 receptor peptide agonists have unsatisfactory potency, selectivity, and stability profiles that hinder their clinical viability. Furthermore, many of these peptides are not commercial candidates due to stability issues of the peptides in formulation, and the short half-life of such peptides in vivo. Additionally, some VPAC2 receptor peptide agonists have been identified to be inactivated by dipeptidyl peptidase (DPP-IV). Short serum half-lives can hamper the utility of such agonists as therapeutic agents. New treatments are therefore needed that overcome the difficulties associated with current NIDDM drugs.

The present invention seeks to provide improved compounds which are selective for the VPAC2 receptor and which induce insulin secretion from the pancreas only in the presence of high blood glucose levels. The compounds of the present invention are peptides believed to also improve beta cell function. Such peptides may have the physiological effect of inducing insulin secretion without causing gastrointestinal (GI) side effects or a corresponding increase in hepatic glucose excretion, and generally have enhanced selection compared to known VPAC2 receptor peptide agonists properties, potency and/or in vivo peptide stability.

According to a first aspect of the present invention, there is provided a VPAC2 receptor peptide agonist comprising a sequence of the following formula:

Xaa _{1 -Xaa 2} -Xaa ₃ -Xaa ₄ -Xaa ₅ -Xaa ₆ -Thr-Xaa 8 -Xaa ₉ -Xaa 10 -Thr-Xaa _{12 -Xaa 13} -Xaa ₁₄ -Xaa ₁₅ -Xaa ₁₆ -Xaa ₁₇ -Xaa ₁₈ -Xaa ₁₉ -Xaa ₂₀ -Xaa ₂₁ -Xaa ₂₂ -Xaa ₂₃ -Xaa ₂₄ -Xaa ₂₅ -Xaa 26 -Xaa ₂₇ -Xaa ₂₈ -Xaa ₂₉ -Xaa ₃₀ -Xaa _{31 -Xaa 32}

Formula 1 (SEQ ID NO: 1)

in:

Xaa ₁ is His, dH or absent;

Xaa ₂ is dA, Ser, Val, Gly, Thr, Leu, dS, Pro or Aib;

Xaa ₃ is Asp or Glu;

Xaa ₄ is Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib or NMeA;

Xaa ₅ is Val, Leu, Phe, Ile, Thr, Trp, Tyr, dV, Aib or NMeV;

Xaa ₆ is Phe, Ile, Leu, Thr, Val, Trp or Tyr;

Xaa ₈ is Asp, Glu, Ala, Lys, Leu, Arg or Tyr;

Xaa ₉ is Asn, Gln, Glu, Ser, Cys or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₀ is Tyr, Trp or Tyr(OMe);

Xaa ₁₂ is Arg, Lys, hR, Orn, Aib, Ala, Leu, Gln, Phe or Cys;

Xaa ₁₃ is Leu, Phe, Glu, Ala, Aib, Ser, Cys or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₄ is Arg, Leu, Lys, Ala, hR, Orn, Phe, Gln, Aib or Cit;

Xaa ₁₅ is Lys, Ala, Arg, Glu, Leu, Orn, Phe, Gln, Aib, K(Ac), Cys, K(W) or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₆ is Gln, Lys, Ala, Ser, Cys or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₇ is Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W);

Xaa ₁₈ is Ala, Ser, Cys or Abu;

Xaa ₁₉ is Ala, Leu, Gly, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or Abu;

Xaa ₂₀ is Lys, Gln, hR, Arg, Ser, Orn, Ala, Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cys or K(CO( _CH2 ) _2SH ) ;

Xaa ₂₁ is Lys, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Ser, Cys, K(W), K(CO( _CH2 ) _2SH ) or hC;

Xaa ₂₂ is Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib or Ser;

Xaa ₂₃ is Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib or Ser;

Xaa ₂₄ is Gln, Asn, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or K(W);

Xaa ₂₅ is Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr, Aib, Glu, Cys, K(CO( _CH2 ) _2SH ) or hC;

Xaa ₂₆ is Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W);

Xaa ₂₇ is Lys, hR, Arg, Gln, Orn or dK;

Xaa ₂₈ is Asn, Gln, Lys, Arg, Aib, Orn, hR, Pro, dK, Cys, K(CO( _CH2 ) _2SH ) or K(W);

Xaa ₂₉ is Lys, Ser, Arg, Asn, hR, Cys, Orn or absent;

Xaa ₃₀ is Arg, Lys, Ile, hR or absent;

Xaa ₃₁ is Tyr, His, Phe, Gln or absent; and

Xaa ₃₂ is Cys or absent;

and satisfies that if Xaa ₂₉ , Xaa ₃₀ , Xaa ₃₁ or Xaa ₃₂ are absent, the next amino acid present downstream is the next amino acid in the sequence of the peptide agonist;

And, the agonist comprises a C-terminal extension comprising the following amino acid sequence:

GGPSSGAPPPK (EC ₁₆ ) (SEQ ID NO: 8)

Wherein, the C-terminal amino acid may be amidated.

Preferably, the VPAC2 receptor peptide agonist comprises a sequence of the following formula:

His-Ser-Xaa ₃ -Ala-Val-Phe-Thr-Xaa ₈ -Xaa ₉ -Xaa ₁₀ -Thr-Xaa ₁₂ -Xaa ₁₃ -Xaa ₁₄ -Xaa ₁₅ -Xaa 16 -Xaa ₁₇ -Xaa _{18 -Xaa 19} - Xaa ₂₀ -Xaa ₂₁ -Xaa ₂₂ -Xaa ₂₃ -Xaa ₂₄ -Xaa ₂₅ -Xaa ₂₆ -Xaa ₂₇ -Xaa ₂₈ -Xaa ₂₉ -Xaa ₃₀ -Xaa ₃₁ -Xaa ₃₂

Formula 2 (SEQ ID NO: 2)

in:

Xaa ₃ is Asp or Glu;

Xaa ₈ is Asp, Glu, Ala, Lys, Leu, Arg or Tyr;

Xaa ₉ is Asn, Gln, Glu, Ser, Cys or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₀ is Tyr, Trp or Tyr(OMe);

Xaa ₁₂ is Arg, Lys, hR, Orn, Aib, Ala, Leu, Gln, Phe or Cys;

Xaa ₁₃ is Leu, Phe, Glu, AIa, Aib, Ser, Cys or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₄ is Arg, Leu, Lys, Ala, hR, Orn, Phe, Gln, Aib or Cit;

Xaa ₁₅ is Lys, Ala, Arg, Glu, Leu, Orn, Phe, Gln, Aib, K(Ac), Cys, K(W) or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₆ is Gln, Lys, Ala, Ser, Cys or K(CO(CH ₂ ) ₂ SH);

Xaa ₁₇ is Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W);

Xaa ₁₈ is Ala, Ser, Cys or Abu;

Xaa ₁₉ is Ala, Leu, Gly, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or Abu;

Xaa ₂₀ is Lys, Gln, hR, Arg, Ser, Orn, Ala, Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cys or K(CO( _CH2 ) _2SH ) ;

Xaa ₂₁ is Lys, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Ser, Cys, K(W), K(CO( _CH2 ) _2SH ) or hC;

Xaa ₂₂ is Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib or Ser;

Xaa ₂₃ is Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib or Ser;

Xaa ₂₄ is Gln, Asn, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or K(W);

Xaa ₂₅ is Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr, Aib, Glu, Cys, K(CO( _CH2 ) _2SH ) or hC;

Xaa ₂₆ is Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W);

Xaa ₂₇ is Lys, hR, Arg, Gln, Orn or dK;

Xaa ₂₈ is Asn, Gln, Lys, Arg, Aib, Orn, hR, Pro, dK, Cys, K(CO( _CH2 ) _2SH ) or K(W);

Xaa ₂₉ is Lys, Ser, Arg, Asn, hR, Cys, Orn or absent;

Xaa ₃₀ is Arg, Lys, Ile, hR or absent;

Xaa ₃₁ is Tyr, His, Phe, Gln or absent; and

Xaa ₃₂ is Cys or absent;

and satisfies that if Xaa ₂₉ , Xaa ₃₀ , Xaa ₃₁ or Xaa ₃₂ are absent, the next amino acid present downstream is the next amino acid in the sequence of the peptide agonist;

And, the agonist comprises a C-terminal extension comprising the following amino acid sequence:

GGPSSGAPPPK (EC ₁₆ ) (SEQ ID NO: 8)

Wherein, the C-terminal amino acid may be amidated.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₃ is Asp or Glu, and Xaa ₈ is Asp or Glu , Xaa ₉ is Asn or Gln, Xaa ₁₀ is Tyr or Tyr (OMe), Xaa ₁₂ is Arg, hR, Lys or Orn, Xaa ₁₄ is Arg, Gln, Aib, hR, Orn, Cit, Lys, Ala or Leu, Xaa ₁₅ is Lys, Aib, Orn or Arg, Xaa ₁₆ is Gln or Lys, Xaa ₁₇ is Val, Leu, Ala, Ile, Lys or Nle, Xaa ₁₉ is Ala or Abu, Xaa ₂₀ is Lys, Val, Leu, Aib , Ala, Gln or Arg, Xaa ₂₁ is Lys, Aib, Orn, Ala, Gln or Arg, Xaa ₂₃ is Leu or Aib, Xaa ₂₅ is Ser or Aib, Xaa ₂₇ is Lys, Orn, hR or Arg, Xaa ₂₈ is Asn, Gln, Lys, hR, Aib, Orn or Pro and/or Xaa ₂₉ is Lys, Orn, hR or absent.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₈ is Glu, Xaa ₉ is Gln and Xaa ₁₀ is Tyr(OMe).

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of Formula 1 (SEQ ID NO: 1) or Formula 2 (SEQ ID NO: 2), wherein Xaa ₁₄ or Xaa ₁₅ is Aib.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₂₀ or Xaa ₂₁ is Aib.

More preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₁₅ is Aib and/or Xaa ₂₀ is Aib.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₁₂ , Xaa ₂₁ , Xaa ₂₇ and Xaa ₂₈ are all Orn.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₁₉ is Abu.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₂₃ is Aib.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₂₅ is Aib. .

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises the sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein Xaa ₃₀ , Xaa ₃₁ and Xaa ₃₂ are absent. Even more preferably, Xaa ₂₉ , Xaa ₃₀ , Xaa ₃₁ and Xaa ₃₂ are all absent.

Preferably, the VPAC2 receptor peptide agonist of the present invention is polyethylene glycol added.

A PEG molecule may be covalently linked to any Lys, Cys, K(W) or K(CO( _CH2 ) _2SH ) residue anywhere on the VPAC2 receptor peptide agonist according to the first aspect of the invention.

Any Lys residue in the VPAC2 receptor peptide agonist may be substituted with K(W) or K(CO( _CH2 ) _2SH ), which may be polyethylene glycol added. In addition, any Cys residues in the peptide agonist may be substituted with a modified cysteine residue, eg hC. Modified Cys residues can be covalently attached to PEG molecules.

When there is more than one PEG molecule, there may be combinations of Lys, Cys, K(CO( _CH2 ) _2SH ) and K(W) added to polyethylene glycol. For example, if there are two PEG molecules, one can be linked to a Lys residue and one can be linked to a Cys residue.

Preferably, the PEG molecule is branched. Alternatively, the PEG molecule can be linear.

Preferably, the molecular weight of the PEG molecule is between 1000 Daltons and 100,000 Daltons. More preferably, the PEG molecule is selected from 10,000, 20,000, 30,000, 40,000, 50,000 and 60,000 Daltons. Even more preferably, it is selected from 20,000, 30,000, 40,000 or 60,000 Daltons. When there are two PEG molecules covalently linked to the peptide agonists of the invention, each of 1,000 to 40,000 Daltons and preferably, have molecular weights of 20,000 and 20,000 Daltons, 10,000 and 30,000 Daltons, 30,000 and 30,000 Daltons, or 20,000 and 40,000 Daltons.

Preferably, the VPAC2 receptor peptide agonists of the invention are cyclic.

VPAC2 receptor peptide agonists can be cyclized via a lactam bridge. Preferably, the lactam bridge is formed by covalently bonding the side chain of the residue on Xaa _n to the side chain of the residue on Xaa _n+4 , where n is 1 to 28. Preferably, n is 12, 20 or 21. More preferably, n is 21. Also preferably, the lactam bridge is formed by covalent attachment of Lys or Orn residue side chains to Asp or Glu residue side chains. Lys or Orn residues may be replaced by Dab residues whose side chains may be covalently linked to the side chains of Asp or Glu residues.

Alternatively, VPAC2 receptor peptide agonists can also be cyclized via disulfide bonds. Preferably, the disulfide bond is formed by covalently linking the side chain of the residue on Xaa _n to the side chain of the residue on Xaa _n+4 , wherein n is 1 to 28. Preferably, n is 12, 20 or 21. More preferably, n is 21. Also preferably, the disulfide bond is formed by a covalent linkage between a Cys or hC residue side chain and another Cys or hC residue side chain.

Alternatively, a lactam bridge or disulfide bond can be formed by covalently linking the side chain of the residue on Xaa _n to the side chain of the residue on Xaa _n+3 , where n is 1-28. Lactam bridges or disulfide bridges, where i is 1 to 24, can also be formed by covalently linking the side chains of residues on Xaa _i to the side chains of residues on Xaa _i+7 or Xaa _i+8 .

The VPAC2 receptor peptide agonist sequence may also contain a histidine residue at the N-terminus of the peptide preceding _Xaa1 .

Preferably, the VPAC2 receptor peptide agonist according to the first aspect of the present invention further comprises an N-terminal modification at the N-terminus of the peptide agonist, wherein the N-terminal modification is selected from:

(a) adding D-histidine, isoleucine, methionine or norleucine;

(b) adding a peptide containing the sequence Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg (SEQ ID NO: 6), wherein Arg is linked to the N-terminus of the peptide agonist;

(c) Adding C ₁ -C ₁₆ alkyl optionally with one or more substituents independently selected from aryl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ elect to replace

(d) Adding -C(O)R ¹ , wherein R ¹ is independently selected from aryl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen, -SH and -CF ₃ or multiple substituents optionally substituted C ₁ -C ₁₆ alkyl; independently selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ - Aryl optionally substituted by one or more substituents of C ₆ alkoxy, -NH ₂ , -OH, halogen, and -CF ₃ ; independently selected from C ₁ -C ₆ alkyl, C ₂ -C One or more substituents of ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ optionally substituted aryl C ₁ - C ₄ alkyl; -NR ² R ³ , wherein R ² and R ³ are independently hydrogen, C ₁ -C ₆ alkyl, aryl or arylC ₁ -C ₄ alkyl; -OR ⁴ , wherein R ⁴ is _{C 1} -C ₁₆ alkyl optionally substituted with one or more substituents independently selected from aryl, C 1 -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ , independently selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ Optionally substituted aryl with one or more substituents independently selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkane One or more substituents of oxy, -NH ₂ , -OH, halogen and -CF ₃ optionally substituted aryl C ₁ -C ₄ alkyl; or 5-pyrrolidin-2-one;

(e) adding -SO ₂ R ⁵ , wherein R ⁵ is aryl, aryl C ₁ -C ₄ alkyl or C ₁ -C ₁₆ alkyl;

(f) forming a succinimide group optionally substituted with C ₁ -C ₆ alkyl or -SR ⁶ , wherein R ⁶ is hydrogen or C ₁ -C ₆ alkyl;

(g) adding methionine sulfoxide;

(h) adding biotinyl-6-aminocaproic acid (6-aminocarproicacid); and

(i) Add -C(=NH)-NH ₂ .

Preferably, the N-terminal modification is the addition of a group selected from the group consisting of acetyl, propionyl, butyryl, valeryl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenyl Acetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminocaproic acid, and -C(=NH) _-NH2 . A particularly preferred N-terminal modification is the addition of an acetyl or hexanoyl group.

Those skilled in the art will understand that VPAC2 receptor peptide agonists comprising various combinations of peptide sequences according to Formula 1 or Formula 2 described herein and N-terminal modifications can be produced based on the above disclosure.

Preferably, the VPAC2 receptor peptide agonist according to the first aspect of the present invention comprises the following amino acid sequence:

Agonist# SEQ ID NO sequence P603 7 C6-HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQAibIOrnOrnGGPSSGAPPPK(E-C16)-NH ₂

According to the second aspect of the present invention, there is provided a pharmaceutical composition comprising the cyclic VPAC2 receptor peptide agonist used in the present invention and one or more pharmaceutically acceptable diluents, carriers and/or excipients.

According to a third aspect of the present invention there is provided a VPAC2 receptor peptide agonist of the present invention for use as a medicament.

According to a fourth aspect of the present invention there is provided a VPAC2 receptor peptide agonist of the present invention for use in the treatment of non-insulin-dependent diabetes or insulin-dependent diabetes or for the suppression of food intake.

According to a fifth aspect of the present invention, there is provided the use of the VPAC2 receptor peptide agonist of the present invention for the manufacture of a medicament for treating non-insulin-dependent diabetes or insulin-dependent diabetes or for curbing food ingest.

According to another aspect of the present invention, there is provided a method of treating non-insulin-dependent diabetes or insulin-dependent diabetes or suppressing food intake in a patient in need thereof, said method comprising administering an effective amount of a VPAC2 receptor peptide of the present invention agonist.

According to yet another aspect of the present invention, there is provided a pharmaceutical composition containing the VPAC2 receptor peptide agonist of the present invention, said composition being used for treating non-insulin-dependent diabetes or insulin-dependent diabetes or for suppressing food intake.

The VPAC2 receptor peptide agonists of the present invention have the advantage that they have enhanced selectivity, potency and/or stability relative to known VPAC2 receptor peptide agonists. In vivo, the C-terminal palmitic acid group may bind serum albumin, thereby preventing renal filtration and prolonging the biological effects of VPAC2 receptor peptide agonists.

The VPAC2 receptor peptide agonist of the present invention may be added with polyethylene glycol.

Covalent attachment of one or more PEG molecules to specific residues of the VPAC2 receptor peptide agonist results in an increased half-life and reduced clearance compared to VPAC2 receptor peptide agonists without added PEG Biologically active VPAC2 receptor peptide agonist added with polyethylene glycol.

The VPAC2 receptor peptide agonists of the invention may be cyclic.

Cyclic VPAC2 receptor peptide agonists have restricted conformational mobility compared to small/medium sized linear VPAC2 peptide receptor agonists, thus cyclic peptides have less allowable Conformation. Restricting the conformational flexibility of linear peptides by cyclization results in enhanced receptor binding affinity, increased selectivity, and improved proteolytic stability and bioavailability compared to linear peptides.

The term "VPAC2" is used to refer to a specific receptor (Lutz et al., FEBS Lett., 458:197-203 (1999); Adamou et al., Biochem.Biophys.Res.Commun., 209:385-392 (1995 )), which can be activated by the agonists of the present invention. The term is also used to refer to the agonists of the present invention.

A "selective VPAC2 receptor peptide agonist" or "VPAC2 receptor peptide agonist" of the present invention is a peptide that selectively activates the VPAC2 receptor to induce insulin secretion. Preferably, the sequence of the selective VPAC2 receptor peptide agonist of the invention has 28 to 32 naturally occurring and/or non-naturally occurring amino acids, and additionally comprises a C-terminal extension comprising the following amino acid sequence: GGPSSGAPPPK( EC ₁₆ ).

"VPAC2 receptor peptide agonist selectively added to polyethylene glycol" or "VPAC2 receptor peptide agonist added to polyethylene glycol" is a combination of one or more polyethylene glycol (PEG) molecules or derivatives thereof A valency-linked selective VPAC2 receptor peptide agonist wherein each PEG is linked to a cysteine or lysine amino acid, or to a K(W) or K(CO(CH ₂ ) ₂ SH) residue.

A "selective cyclic VPAC2 receptor peptide agonist" or "cyclic VPAC2 receptor peptide agonist" is a selective VPAC2 receptor that cyclizes by a covalent linkage between the side chains of two amino acids in the peptide chain Peptide agonists. Covalent bonds may for example be lactam bridges or disulfide bonds.

The selective VPAC2 receptor peptide agonists of the invention have a C-terminal extension. The C-terminal extension of the present invention comprises the sequence GGPSSGAPPPK (EC ₁₆ ), and at the N-terminus of said C-terminal extension is linked to formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2) by a peptide bond The peptide sequences are connected at the C-terminus. The sequence GGPSSGAPPPK (E-C16) is a variant of the C-terminal sequence of exendin-4. The C-terminal lysine residue has attached to its side chain a glutamic acid residue acylated at the α-amino group with palmitic acid.

As used herein, the term "linked" when referring to the term C-terminal extension includes direct addition or attachment of amino acids or chemical groups to the C-terminus of the peptide sequence of Formula 1 or Formula 2.

Optionally, the selective VPAC2 receptor peptide agonist may also have an N-terminal modification. The term "N-terminal modification" as used herein includes adding or linking amino acids or chemical groups directly at the N-terminal of the peptide, as well as forming chemical groups that bind to the nitrogen at the N-terminal of the peptide.

N-terminal modifications may include the addition of one or more naturally occurring or non-naturally occurring amino acids to the VPAC2 receptor peptide agonist peptide sequence, preferably no more than 10 amino acids, with one amino acid being more preferred. Naturally occurring amino acids that can be added to the N-terminus include methionine and isoleucine. The modified amino acid added to the N-terminus may be D-histidine. Alternatively, the following amino acids can be added to the N-terminus: SEQ ID NO: 6Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg, wherein Arg is linked to the N-terminus of the peptide agonist. Preferably, any amino acids added to the N-terminus are linked to the N-terminus by a peptide bond.

The term "linked" as used herein with respect to N-terminal modification includes directly adding or linking amino acids or chemical groups at the N-terminal of the VPAC2 receptor agonist. Addition of the above-mentioned N-terminal modifications can be achieved under conventional coupling conditions for peptide bond formation.

The N-terminus of the peptide agonist can also be modified by the addition of an alkyl group (R), preferably a C ₁ -C ₁₆ alkyl group, forming (R)NH-.

Alternatively, the N-terminus of the peptide agonist can be modified by adding a set of amides of formula -C(O) ^R1 to form ^R1C (O)NH-. Addition of a set of formula -C(O)R ¹ can be achieved by reaction with an organic acid of formula R ¹ COOH. The prior art has demonstrated the use of acylation to modify the N-terminus of amino acid sequences (eg, Gozes et al., J. Pharmacol Exp Ther, 273:161-167 (1995)). Addition of a set of formula -C(O)R ¹ can result in the formation of a urea group (see WO01/23240, WO2004/006839) or a carbamate group at the N-terminus. In addition, the N-terminus can be modified by adding pyroglutamic acid, or 6-aminocaproic acid.

The N-terminus ^of a peptide agonist can be modified by adding a set of formula _-SO2R5 to form a sulfonamide group at the N-terminus.

The N-terminus of the peptide agonist can also be modified by reaction with succinic anhydride to form a succinimide group at the N-terminus. Succinimide binds to the nitrogen at the N-terminus of the peptide.

The N-terminus can be optionally modified by addition of methionine sulfoxide, biotinyl-6-aminocaproic acid, or -C(=NH) _-NH2 . The addition of -C(=NH)-NH ₂ is a guanidation modification, changing the terminal NH ₂ of the N-terminal amino acid to -NH-C(=NH)-NH ₂ .

Most sequences of the invention, including N-terminal modifications and C-terminal extensions, contain standard one-letter or three-letter codes for the 20 naturally occurring amino acids. The other codes used are defined as follows:

Ac = acetyl

C6 = hexanoyl

d = (non-naturally occurring) D-isoform of each amino acid

For example, dA = D-alanine, dS = D-serine, dK = D-lysine

hR = homoarginine

_ = unoccupied position

Aib = aminoisobutyric acid

CH ₂ = methylene

OMe = Methoxy

Nle = norleucine

NMe = N-methyl group attached to alpha amino group of amino acid,

For example, NMeA = N-methylalanine, NMeV = N-methylvaline

Orn = ornithine

K(CO(CH ₂ ) ₂ SH)=ε-(3'-mercaptopropionyl)-lysine

K(W)＝ε-(L-tryptophanyl)-lysine

Abu = α-amino-n-butyric acid (butyric acid) or 2-aminobutyric acid (aminobutanoic acid)

Cit = citrulline

Dab = diaminobutyric acid (diaminobutyric acid)

K(Ac)＝ε-acetyllysine

PEG = polyethylene glycol

PEG40K = PEG molecule of 40,000 Daltons

PEG30K = PEG molecule of 30,000 Daltons

PEG20K = PEG molecule of 20,000 Daltons

K(EC ₁₆ )=(ε-(γ-L-glutamyl(N-α-palmitoyl))-lysine

＝内酰胺桥或二硫键

= lactam bridge or disulfide bridge

VIP occurs naturally as a single sequence of 28 amino acids. But PACAP exists as a 38 amino acid peptide (PACAP-38) or a 27 amino acid peptide (PACAP-27) with amidated carboxyl groups (Miyata et al., Biochem Biophys Res Commun, 170:643-648 (1990)) . The sequences of VIP, PACAP-27 and PACAP-38 are shown below:

peptide Seq.ID# sequence VIP SEQ ID NO: 3 HSDAVFTDNYTRLRKQMAVKKYLNSILN

PACAP-27 SEQ ID NO: 4 HSDGIFTDSYSRYRKQMAVKKYLAAVL-NH ₂ PACAP-38 SEQ ID NO: 5 HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYQRVKNK-NH ₂

The term "naturally occurring amino acid" as used herein means the 20 amino acids encoded by the human genetic code (ie, the 20 standard amino acids). The above 20 amino acids are: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine , lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Examples of "non-naturally occurring amino acids" include synthetic amino acids and amino acids modified by the body. These include D-amino acids, arginine-like amino acids (e.g., homoarginine), and other amino acids with additional methylene groups in the side chains ("homo" amino acids), as well as modified amino acids (e.g., norleucine, Lysine (isopropyl) - where the side chain amine of lysine is modified with isopropyl). Amino acids such as ornithine, aminoisobutyric acid, and GABA are also included.

As used herein, "selectivity" means that a VPAC2 receptor peptide agonist has increased selectivity for the VPAC2 receptor compared to other known receptors. The degree of selectivity is determined by the ratio of VPAC2 receptor binding affinity to VPAC1 receptor binding affinity, and by the ratio of VPAC2 receptor binding affinity to PAC1 receptor binding affinity. Determination of binding affinity is described in Example 4 below.

"Insulinotropic activity" refers to the ability to stimulate insulin secretion in response to elevated glucose levels, resulting in cellular uptake of glucose and a reduction in plasma glucose levels. Insulinotropic activity can be assessed by methods known in the art, including by measuring VPAC2 receptor binding activity or receptor activation (e.g., insulin secretion by insulinoma cell lines or islets, intravenous glucose tolerance test (IVGTT), intraperitoneal glucose tolerance Test (IPGTT) and Oral Glucose Tolerance Test (OGTT)). Routine measurement of insulinotropic activity in humans is by measuring insulin levels or C-peptide levels. The selective VPAC2 receptor peptide agonists of the present invention have insulinotropic activity.

"In vitro potency" as used herein is a measure of the ability of a peptide to activate the VPAC2 receptor in a cell-based assay. In vitro potency, expressed as " _EC50 ", is the effective concentration of compound that results in a 50% maximal increase in activity in a single dose response assay. For the purposes of the present invention, in vitro potency was determined using two different assays: DiscoverRx and Alpha Screen. See Examples 3 and 5 for additional details of the above assays. Whilst the above assays were performed in different ways, the results demonstrated a general correlation between the two assays.

The term "plasma half-life" refers to the time at which half of the molecule of interest previously circulating in the plasma is cleared. An alternative term used is "elimination half-life". The terms "prolonged" or "longer" as used in reference to plasma half-life or elimination half-life refer to a peptide in a form without added polyethylene glycol or a native peptide as determined under comparable conditions. ) compared to VPAC2 receptor peptide agonists added with polyethylene glycol had a statistically significant increase in half-life. The half-life reported here is the elimination half-life; it corresponds to the terminal log-linear rate of elimination. Those skilled in the art know that half-life is a derived parameter that varies as a function of clearance and volume of distribution.

Clearance is a measure of the body's ability to get rid of a drug. When clearance is reduced due to, for example, drug modification, half-life is expected to be increased. However, this reciprocal relationship is exact only if the volume of distribution does not change. The equation t _1/2 ≈0.693(V/C) gives an effective approximate relationship between terminal log-linear half-life (t _1/2 ), clearance (C) and volume distribution (V). Clearance does not indicate how much drug is removed, but rather the volume of biological fluid, such as blood or plasma, that must be completely freed of drug in order to achieve elimination. Clearance is expressed as volume per unit time.

As used herein, "percent (%) sequence identity" is used to denote sequences that have similar (identical or conservatively substituted) amino acids at similar positions or regions when aligned, wherein the identical or conservatively substituted amino acids are those that do not alter the activity or function of the protein compared to the starting protein. For example, two amino acid sequences that are at least 85% identical to each other have at least 85% similarity (identical or conservatively substituted residues) at similar positions, wherein up to 3 gaps are optimally allowed when aligned, provided that With regard to gaps, a total of no more than 15 amino acid residues were affected.

The reference peptides used herein to calculate percent sequence identity are:

P603 C6-HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQAibIOrnOrnGGPSSGAPPPK(E-C16)-NH ₂

Percent sequence identity can be calculated by determining the number of residues that differ between a peptide encompassed by the invention and a reference peptide such as P603 (SEQ ID NO: 7), taking this number and dividing it by the number of amino acids of the reference peptide (for example, P603 is 39 amino acids), multiply the result by one hundred, and subtract the result from 100. For example, a sequence of 39 amino acids that differs from P603 by 4 amino acids has a percent (%) sequence identity of 90% (eg, 100-((4/39)×100)). For sequences exceeding 39 amino acids, the number of residues that differ from the P603 sequence will include, for the purposes of the calculations above, the additional amino acids above 39. For example, a sequence of 40 amino acids, of which 4 amino acids differ from 39 amino acids in the P603 sequence, and an additional amino acid at the carboxy terminus that is not present in the P603 sequence, differs from P603 by a total of 5 amino acids. Thus, the sequences have a percent (%) sequence identity of 87% (eg, 100-((5/39)×100)). The degree of sequence identity can be determined using methods generally known in the art (see, e.g., Wilbur, W.J. and Lipman, D.J.Proc. Natl. Acad. Sci. USA 80:726-730 (1983) and Myers E. and Miller W ., Comput. Appl. Biosci. 4:11-17 (1988)). A program that can be used to determine the degree of similarity is the one pair (one pair) method of MegAlign Lipman-Pearson (using default parameters), which is available from DNAstar Corporation, 1128, Selfpark Street, Madison, Wisconsin, 53715, USA as a Lasergene system. partially obtained. Another program that can be used is Clustal W. This is a multiple sequence alignment software package for DNA or protein sequences developed by Thompson et al. (Nucleic Acids Research, 22(22):4673-4680 (1994)). This tool is effective for performing cross-species comparisons of related sequences and observing sequence conservation. Clustal W is a general purpose multiple sequence alignment program for DNA or proteins. It produces biologically meaningful multiple sequence alignments of distinct sequences. It calculates the best matches for selected sequences, aligning them so that identities, similarities and differences can be seen. Evolutionary relationships can be seen by looking at Cladograms or Phylograms.

The sequence of the selective VPAC2 receptor peptide agonist of the present invention is selectively used for the VPAC2 receptor, and preferably has a ratio of 60% to 70%, 60% to 65%, 65% to P603 (SEQ ID NO: 7) to 70%, 70% to 80%, 70% to 75%, 75% to 80%, 80% to 90%, 80% to 85%, 85% to 90%, 90% to 97%, 90% to 95% % or in the range of 95% to 97% sequence identity. Preferably, the sequence has greater than 82% sequence identity to P603 (SEQ ID NO: 7). More preferably, the sequence has greater than 90% sequence identity to P603 (SEQ ID NO: 7). Even more preferably, the sequence has greater than 92% sequence identity to P603 (SEQ ID NO: 7). Still more preferably, the sequence has greater than 95% sequence identity or 97% sequence identity to P603 (SEQ ID NO: 7).

The term "C ₁ -C ₁₆ alkyl" as used herein means a monovalent saturated linear, branched or cyclic hydrocarbon group having 1 to 16 carbon atoms, or when cyclic, 3 to 16 carbons atom. Thus, the term "C ₁ -C ₁₆ alkyl" includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-heptyl, n- Octyl, Cyclopropyl, Cyclobutyl, Cyclopentyl and Cyclohexyl. C ₁ -C ₁₆ alkyl groups may be optionally substituted with one or more substituents including, for example, aryl, C ₁ -C ₆ alkoxy, -OH, halogen, -CF ₃ and - sh.

The term "C ₁ -C ₆ alkyl" as used herein means a monovalent saturated linear, branched or cyclic hydrocarbon group having 1 to 6 carbon atoms or 3 to 6 carbon atoms when cyclic. Thus, the term "C ₁ -C ₆ alkyl" includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclopropyl, Butyl, cyclopentyl and cyclohexyl. A C ₁ -C ₆ alkyl group may be optionally substituted with one or more substituents.

The term "C ₂ -C ₆ alkenyl" as used herein means a monovalent linear, branched or cyclic hydrocarbon group having at least one double bond and 2 to 6 carbon atoms or, when cyclic, 3 to 6 carbon atoms. 6 carbon atoms. Thus, the term " _C2 - _C6 alkenyl" includes vinyl, prop-2-enyl, but-3-enyl, pent-4-enyl and isopropenyl. C ₂ -C ₆ alkenyl may be optionally substituted with one or more substituents.

The term "C ₂ -C ₆ alkynyl" as used herein means a monovalent straight or branched chain hydrocarbon group having at least one triple bond and 2 to 6 carbon atoms. Thus, the term " _C2 - _C6 alkynyl" includes prop-2-ynyl, but-3-ynyl and pent-4-ynyl. A C ₂ -C ₆ alkynyl group may be optionally substituted with one or more substituents.

The term "C ₁ -C ₆ alkoxy" as used herein means a monovalent unsubstituted saturated linear or branched hydrocarbon group having 1 to 6 carbon atoms attached to a substitution point of a divalent O group. Thus, the term "C ₁ -C ₆ alkoxy" includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert- butoxyl. A C ₁ -C ₆ alkoxy group may be optionally substituted with one or more substituents.

The term "halo" or "halogen" means fluorine, chlorine, bromine or iodine.

When used alone or as part of a group, the term "aryl" is a 5 to 10 membered aromatic or heteroaromatic group which includes phenyl, a 5 or 6-membered monocyclic aromatic heterocyclic group (wherein each member can be optionally substituted by 1, 2, 3, 4 or 5 substituents (depending on the number of substitution positions available)), naphthyl or 8-, 9- or 10-membered bicyclic aromatic hetero Cyclic groups (wherein each member may be optionally substituted with 1, 2, 3, 4, 5 or 6 substituents (depending on the number of available substitution positions)). In the definition of aryl, suitable substituents include C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, amino, hydroxy, halogen, -SH and CF ₃ .

The term "aryl C ₁ -C ₄ alkyl" as used herein means a C ₁ -C ₄ alkyl substituted with an aryl group. Thus, the term "aryl C ₁ -C ₄ alkyl" includes benzyl, 1-phenethyl (α-methylbenzyl), 2-phenethyl, 1-naphthylmethyl or 2-naphthylmethyl.

The term "naphthyl" includes 1-naphthyl and 2-naphthyl. 1-Naphthyl is preferred.

The term "benzyl" as used herein means a monovalent unsubstituted phenyl group attached to the point of substitution through a _-CH2- group.

The term "5- or 6-membered monocyclic aromatic heterocyclic group" as used herein means a monocyclic aromatic group with a total of 5 or 6 atoms in the ring, wherein 1 to 4 of the above-mentioned atoms are independently selected from N, O and S. Preferred groups have 1 or 2 atoms in the ring each independently selected from N, O and S. Examples of five-membered monocyclic aromatic heterocyclic groups include pyrrolyl (also known as azolyl), furanyl (furanyl), thienyl, pyrazolyl (also known as 1H-pyrazolyl and 1,2-diazolyl), imidazolyl , oxazolyl (also known as 1,3-oxazolyl), isoxazolyl (also known as 1,2-oxazolyl), thiazolyl (also known as 1,3-thiazolyl), isothiazolyl (also known as Called 1,2-thiazolyl), triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl and thiatriazolyl. Examples of the 6-membered monocyclic aromatic heterocyclic group include pyridyl, pyrimidyl, pyrazinyl, pyridazinyl and triazinyl.

The term "8-, 9- or 10-membered bicyclic aromatic heterocyclic group" as used herein means a fused bicyclic aromatic group having a total of 8, 9 or 10 atoms in the ring system, wherein 1 to 4 of the above atoms are independently is selected from N, O and S. Preferred groups have 1 to 3 atoms in the ring system, each independently selected from N, O and S. Suitable 8-membered bicyclic aromatic heterocyclic groups include imidazo[2,1-b][1,3]thiazolyl, thieno[3,2-b]thienyl, thieno[2,3-d] [1,3]thiazolyl and thieno[2,3-d]imidazolyl. Suitable 9-membered bicyclic aromatic heterocyclic groups include indolyl, isoindolyl, benzofuryl (also known as benzo[b]furyl), isobenzofuryl (also known as benzo[c]furyl), furyl), benzothienyl (also known as benzo[b]thienyl), isobenzothienyl (also known as benzo[c]thienyl), indazolyl, benzimidazolyl, 1,3-benzene And oxazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,3 benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzene Isothiazolyl, benzotriazolyl, 1,2,3-benzoxadiazolyl, 2,1,3-benzoxadiazolyl, 1,2,3-benzothiadiazolyl, 2,1,3-Benzothidiazolyl, thienopyridyl, purinyl and imidazo[1,2-a]pyridine. Suitable 10-membered bicyclic aromatic heterocyclic groups include quinolinyl, isoquinolinyl, cinnoline, quinazolinyl, quinoxalinyl, 1,5-naphthyridyl, 1,6-naphthalene pyridyl, 1,7-naphthyridyl and 1,8-naphthyridyl.

The term "PEG" as used herein means a polyethylene glycol molecule. The typical _{morphology of PEG is a linear polymer with terminal hydroxyl groups and has the formula HO-CH2CH2- ( CH2CH2O} ) _n - _{CH2CH2 -} OH, where n is from about 8 to about 4000. Terminal hydrogens may be replaced with protecting groups such as alkyl or alkanol groups. Preferably, PEG has at least one hydroxyl group, more preferably, it is a terminal hydroxyl group. It is this hydroxyl group that is preferably activated to react with the peptide. There are various forms of PEG that can be used in the present invention. A wide variety of PEG derivatives exist in the art that are suitable for use in the present invention. (See eg, US Patent Nos.: 5445090; 5900461; 5932462; 6436386; 6448369; 6437025; 6448369; 6495659; 6515100 and 6514491, and Zalipsky, S. Bioconjugate Chem. 6:150-165, 1995). The PEG molecule to which the VPAC2 receptor peptide agonist is covalently linked in the present invention is not intended to be limited to a particular type. The molecular weight of the PEG molecule is preferably 500-100,000 Daltons. PEG can be linear or branched and PEGylated VPAC2 receptor peptide agonists can have one, two or three PEG molecules attached to the peptide. More preferably there is one or two PEG molecules per PEGylated VPAC2 receptor peptide agonist, however, when there is more than one PEG molecule per peptide molecule, preferably no more than three. It is further contemplated that both ends of the PEG molecule can be homo- or hetero-functionalized for cross-linking two or more VPAC2 receptor peptide agonists together. When two PEG molecules are present, the PEG molecules are preferably each 20,000 Dalton PEG molecule or each 30,000 Dalton molecule. However, PEG molecules with different molecular weights can also be used, for example, one 10,000 Dalton PEG molecule and one 30,000 Dalton PEG molecule, or one 20,000 Dalton PEG molecule and one 40,000 Dalton PEG molecule.

PEG molecules can be covalently linked to Cys or Lys residues. The PEG molecule can also be covalently linked to a Trp residue coupled to the side chain (K(W)) of a Lys residue. Alternatively, K(CO(CH ₂ ) ₂ SH) groups can be added to polyethylene glycol to form K(CO(CH ₂ ) ₂ S-PEG). Any Lys residue in the peptide agonist can be substituted by K(W) or K(CO( _CH2 ) _2SH ), which in turn can be added to polyethylene glycol. In addition, any Cys residues in the peptide agonist may be replaced by modified cysteine residues, eg hC. Modified Cys residues can be covalently attached to PEG molecules.

As used herein, the term "addition of polyethylene glycol" means that one or more PEG molecules as described above are covalently attached to the VPAC2 receptor peptide agonist of the present invention.

The term "lactam bridge" as used herein denotes a covalent bond, particularly an amide bond, linking the amino terminus of the side chain of one amino acid in a peptide agonist to the carboxy terminus of the side chain of another amino acid in the peptide agonist. Preferably, the lactam bridge is formed by covalently linking the side chain of the residue on Xaa _n to the side chain of the residue on Xaa _n+4 , where n is 1 to 28. Also preferably, the lactam bridge is formed by a covalent linkage between the amino terminus of the side chain of a Lys or Orn residue and the carboxy terminus of the side chain of an Asp or Glu residue.

The term "disulfide bond" as used herein means a covalent bond connecting a sulfur atom at the end of the side chain of one amino acid in the peptide agonist to a sulfur atom at the end of the side chain of another amino acid in the peptide agonist. Preferably, the disulfide bond is formed by covalently linking the side chain of the residue on Xaa _n to the side chain of the residue on Xaa _n+4 , wherein n is 1 to 28. Also preferably, the disulfide bond is formed by a covalent linkage between the side chain of a Cys or hC residue and the side chain of another Cys or hC residue.

According to a preferred embodiment of the present invention, the following VPAC2 receptor peptide agonist is provided, which comprises the amino acid sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein, Xaa ₃ is Asp or Glu, Xaa ₈ is Asp or Glu, Xaa ₉ is Asn or Gln, Xaa ₁₀ is Tyr or Tyr (OMe), Xaa ₁₂ is Arg, hR, Lys or Orn, Xaa ₁₄ is Arg, Gln, Aib, hR , Orn, Cit, Lys, Ala or Leu, Xaa ₁₅ is Lys, Aib, Orn or Arg, Xaa ₁₆ is Gln or Lys, Xaa ₁₇ is Val, Leu, Ala, Ile, Lys or Nle, Xaa ₁₉ is Ala or Abu , Xaa ₂₀ is Lys, Val, Leu, Aib, Ala, Gln or Arg, Xaa ₂₁ is Lys, Aib, Orn, Ala, Gln or Arg, Xaa ₂₃ is Leu or Aib, Xaa ₂₅ is Ser or Aib, Xaa ₂₇ is Lys, Orn, hR or Arg, Xaa ₂₈ is Asn, Gln, Lys, hR, Aib, Orn or Pro and/or Xaa ₂₉ is Lys, Orn, hR or absent, and comprises a C-terminal extension, said C- The terminal extension included the sequence: GGPSSGAPPPK (EC ₁₆ ), and included an N-terminal modification which was the addition of a hexanoyl or acetyl group.

According to another preferred embodiment of the present invention, the following VPAC2 receptor peptide agonist is provided, which comprises the amino acid sequence of formula 1 (SEQ ID NO: 1) or formula 2 (SEQ ID NO: 2), wherein, Xaa ₈ is Glu, Xaa ₉ is Gln, Xaa ₁₀ is Tyr(OMe), Xaa ₁₂ is Orn, Xaa ₁₅ is Aib, Xaa ₁₉ is Abu, Xaa ₂₀ is Aib, Xaa ₂₁ is Orn, Xaa ₂₃ is Aib, Xaa ₂₅ is Aib, Xaa ₂₇ is Orn and/or Xaa ₂₈ is Orn and comprises a C-terminal extension comprising the sequence: GGPSSGAPPPK (EC ₁₆ ), and an N-terminal modification which is the addition of a hexanoyl or acetyl group.

According to another preferred embodiment of the present invention, the following VPAC2 receptor peptide agonist is provided, which comprises the amino acid sequence of formula 2 (SEQ ID NO: 2), the C-terminal extension comprising the sequence GGPSSGAPPPK (EC ₁₆ ) and N-terminal modification which is the addition of a hexanoyl or acetyl group.

The present invention is based on the discovery that the addition of a C-terminal extension comprising the sequence GGPSSGAPPPK (EC ₁₆ ) to the C-terminus of a peptide sequence according to formula 1 or formula 2 provides features that protect the peptide, as well as enhance activity, selectivity and/or potency. For example, a C-terminal extension can stabilize the helical structure of the peptide, as well as a site near the C-terminus that is prone to enzymatic cleavage. Furthermore, the C-terminally extended peptides disclosed herein can be more selective for the VPAC2 receptor, which can be more potent than VIP, PACAP, and other known VPAC2 receptor peptide agonists.

The PEGylation of proteins can overcome many of the pharmacological and toxicological/immunological problems associated with the use of peptides or proteins as therapeutic agents. However, it is not certain that for any individual peptide, the pegylated form of the peptide will significantly lose biological activity compared to the non-pegylated form of the peptide.

The biological activity of PEGylated proteins can be affected by factors such as: i) the size of the PEG molecule; ii) the specific attachment site; iii) the degree of modification; iv) unfavorable coupling conditions; Whether a linker is used or whether the polymer is directly attached; vi) generation of harmful by-products; vii) damage by activated polymer; or viii) retention of charges. Work carried out on cytokine addition to pegylation demonstrated, for example, the effect that pegylation has. Depending on the conjugation reaction used, polymer modification of cytokines leads to a marked decrease in biological activity [Francis, G.E. et al., (1998) PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimization of coupling techniques, Intl. J .Hem.68:1-18]. Maintaining the biological activity of peptides incorporating polyethylene glycol is even more difficult than that of proteins. Since peptides are smaller than proteins, modification by addition of polyethylene glycol may have a greater effect on biological activity.

The VPAC2 receptor peptide agonists of the present invention can be modified by covalently linking one or more PEG molecules. Peptides incorporated with polyethylene glycol generally have an improved pharmacokinetic profile due to slower proteolytic degradation and renal clearance. Addition of polyethylene glycol will increase the apparent size of VPAC2 receptor peptide agonists, thereby reducing renal filtration and altering biodistribution. The effect of adding polyethylene glycol can protect the epitope of the VPAC2 receptor peptide agonist, thereby reducing the clearance rate of the reticuloendothelium and being recognized by the immune system, and also reducing the degradation of proteolytic enzymes such as DPP-IV.

Covalent attachment of one or more PEG molecules to a small, biologically active VPAC2 receptor peptide agonist poses the risk of negatively affecting the agonist, for example, by destabilizing the intrinsic secondary structure and bioactive conformation and reducing Biological activity, thus making agonists unsuitable as therapeutic agents. Covalent attachment of one or more PEG molecules to specific residues of VPAC2 receptor peptide agonists surprisingly yields bioactive, PEG-added VPAC2 receptor peptide Ethylene glycol has a prolonged half-life and reduced clearance compared to VPAC2 receptor peptide agonists.

To identify potential PEGylated sites of action in VPAC2 receptor peptide agonists, a serine scan can be performed. Ser residues at specific positions in the peptide were substituted, and the potency and selectivity of the Ser-modified peptides were tested. If the Ser substitution has only a minimal effect on potency, and the Ser-modified peptide is chosen for the VPAC2 receptor, then replace the Cys or Lys residue with a Ser residue as a direct or indirect PEGylation site point. Indirect PEGylation of a residue is PEGylation of a chemical group or residue that binds to the residue at the PEGylation site. The indirect PEGylation of Lys includes the PEGylation of K(W) and K(CO(CH ₂ ) ₂ SH).

The invention described herein provides VPAC2 receptor peptide agonists covalently linked to one or more PEGs or derivatives thereof, wherein each PEG can be linked to a Cys or Lys amino acid in the peptide agonist, and K(W) or K(CO(CH ₂ ) ₂ SH) linkage. Addition of polyethylene glycol can enhance the half-life of the selective VPAC2 receptor peptide agonist such that the VPAC2 receptor peptide agonist has an elimination half-life of at least 1 hour, preferably at least 3, 5, 7, 10, 15, 20 or 24 hours, and most preferably, at least 48 hours. The VPAC2 receptor peptide agonist added with polyethylene glycol preferably has a clearance value of 200 ml/h/kg or less, more preferably, 180, 150, 120, 100, 80, 60 ml/h/kg or less, Most preferably, less than 50, 40 or 20 ml/h/kg.

Wild-type VIP has an α-helical structure in the region from aspartic acid at position 8 to isoleucine at position 26. Increasing the helical content of the peptide will enhance potency and selectivity, and at the same time increase protection against enzymatic degradation. The helicity of the peptide can be enhanced using a C-terminal extension. Furthermore, the introduction of a covalent bond linking the side chains of two amino acids on the helical surface, such as a lactam bridge, can also enhance the helicity of the peptide.

It has also been found that modifications to the N-terminus of VPAC2 receptor peptide agonists can enhance potency and/or provide stability against DPP-IV cleavage.

VIP and some known VPAC2 receptor peptide agonists are susceptible to cleavage by various enzymes and thus have a short half-life in vivo. Multiple enzymatic cleavage sites in VPAC2 receptor peptide agonists are discussed below. Cleavage sites are discussed relative to the amino acid positions in VIP (SEQ ID NO: 3), which apply to the sequences indicated herein.

Cleavage of peptide agonists by dipeptidyl peptidase IV (DPP-IV) occurs between position 2 (serine in VIP) and position 3 (aspartic acid in VIP). By adding an N-terminal modification, the agonists of the invention are rendered more stable to DPP-IV cleavage in this region. Examples of N-terminal modifications that may improve stability against DPP-IV cleavage include the addition of acetyl, propionyl, butyryl, valeryl, hexanoyl, methionine, methionine sulfoxide, 3-phenyl Propionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminocaproic acid, and -C(=NH ₂ ) _-NH2 . Preferably, the N-terminal modification is the addition of acetyl or hexanoyl.

There are chymotrypsin cleavage sites between amino acids 10 and 11 (tyrosine and threonine) and 22 and 23 (tyrosine and leucine) of wild-type VIP. Substitutions at positions 10 and/or 11 and positions 22 and/or 23 may increase the stability of the peptide at these positions. For example, substitution of Tyr(OMe) at positions 10 and/or 22 for tyrosine can increase stability. A lactam bridge (eg, linking the side chains of the amino acids at positions 21 and 25) protects positions 22-23 against cleavage.

There is a trypsin cleavage site between amino acids 12 and 13 of wild-type VIP. Some amino acids make the peptide less susceptible to cleavage at this site, for example, when ornithine is at position 12.

In wild-type VIP and many VPAC2 receptor peptide agonists known in the art, there is a cleavage site between the basic amino acids at positions 14 and 15 and between the basic amino acids at positions 20 and 21. Because of the above-mentioned substitutions, the selective VPAC2 receptor peptide agonists of the present invention have improved proteolytic stability in vivo. Preferred substitutions at the aforementioned positions are those that render the peptide less susceptible to cleavage by trypsin-like enzymes, including trypsin. For example, aminoisobutyric acid at position 15, aminoisobutyric acid at position 20, and ornithine at position 21 are all preferred substitutions that can result in improved stability.

There is also a cleavage site between amino acids 25 and 26 of wild-type VIP. This cleavage site can be completely or partially eliminated by substitution of the amino acid at position 25 and/or the amino acid at position 26.

The region of the VPAC2 receptor peptide agonist that includes amino acids at positions 27, 28 and 29 is also susceptible to enzymatic cleavage. Addition of a C-terminal extension may render the peptide agonist more stable against neuroendopeptidase (NEP), which may also increase selectivity against the VPAC2 receptor. This region can also be attacked by trypsin-like enzymes. If this occurs, the peptide agonist may lose its C-terminal extension with additional carboxypeptidase activity, resulting in an inactive form of the peptide. Resistance to cleavage of this region can be increased by substituting ornithine at amino acids 27, 28 and/or 29.

In addition to selective VPAC2 receptor peptide agonists that are resistant to cleavage by various peptidases, the selective VPAC2 receptor peptide agonists of the present invention may also include peptides that are superior to those known in the art Some of the peptides have enhanced selectivity for the VPAC2 receptor, increased potency and/or increased stability.

Preferably, the selective non-polyethylene glycol added VPAC2 receptor peptide agonist has an _EC50 value of less than 2 nM. More preferably, the _EC50 value is less than 1 nM. Even more preferably, the _EC50 is less than 0.5 nM. Still more preferably, the _EC50 value is less than 0.1 nM. Preferably, the selectively pegylated VPAC2 receptor peptide agonist has an _EC50 value of less than 200 nM. More preferably, the _EC50 value is less than 50 nM. Even more preferably, the _EC50 value is less than 30 nM. Even more preferably, the _EC50 value is less than 10 nM.

Example 4 describes the assays used to determine selectivity as the ratio of VPAC2 receptor binding affinity to VPAC1 receptor binding affinity and as the ratio of VPAC2 receptor binding affinity to PAC1 receptor binding affinity and sex ratio to measure. Preferably, the agonist of the invention has a selectivity ratio in which the affinity for the VPAC2 receptor is at least 50 times greater than for the VPAC1 and/or PAC1 receptor. More preferably, the affinity for VPAC2 is at least 100 times greater than for VPAC1 and/or PAC1. Even more preferably, the affinity for VPAC2 is at least 200 times greater than for VPAC1 and/or PAC1. Still more preferably, the affinity for VPAC2 is at least 500 times greater than for VPAC1 and/or PAC1. More preferably, the affinity for VPAC2 is at least 1000 times greater than the ratio for VPAC1 and/or PAC1.

A "selective VPAC2 receptor peptide agonist" as used herein also includes pharmaceutically acceptable salts of the compounds described herein. The selective VPAC2 receptor peptide agonist of the present invention can have enough acidic, enough basic, or bifunctional groups, correspondingly react with any number of inorganic bases, and inorganic and organic acids to form salts. Acids commonly used to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzene-sulfonic acid, etc. acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, trifluoroacetic acid, etc. Examples of such salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, Bromide, Iodide, Acetate, Propionate, Caprate, Caprylate, Acrylate, Formate, Isobutyrate, Hexanoate, Heptanoate, Propiolate, Oxalate , malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, Benzoates, Chlorobenzoates, Methylbenzoates, Dinitrobenzoates, Hydroxybenzoates, Methoxybenzoates, Phthalates, Sulfonates, Xylenesulfonate, Phenylacetate, Phenylpropionate, Phenylbutyrate, Citrate, Lactate, Gamma-Hydroxybutyrate, Glycolate, Tartrate, Methanesulfonate , propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc.

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

The selective VPAC2 receptor peptide agonists of the present invention are preferably formulated as pharmaceutical compositions. Standard pharmaceutical formulation techniques that can be used are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. The selective VPAC2 receptor peptide agonists of the invention may be formulated for administration by buccal, topical, oral, transdermal, nasal or pulmonary routes, or for parenteral administration.

Parenteral administration may include, for example, systemic administration such as by intramuscular, intravenous, subcutaneous, intradermal or intraperitoneal injection. Selective VPAC2 receptor peptide agonists can be administered to a subject in combination with an acceptable pharmaceutical carrier, diluent or excipient as part of a pharmaceutical composition for the treatment of NIDDM or the diseases discussed below. The pharmaceutical composition may be a solution or, if administered parenterally, a suspension of the VPAC2 receptor peptide agonist or a suspension of the VPAC2 receptor peptide agonist complexed with a divalent metal cation such as zinc. Suitable pharmaceutical carriers may contain inert ingredients, which do not interact with the peptide or peptide derivative. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer-lactic acid salt and more. Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol and mannitol.

和Prolease

生物可降解的聚合物也是可以商业获得的。Medisorb

多聚物可以用任意的交酯异构体产生。交酯：乙交酯的比例可以在0:100和100:0之间变动，允许产生宽范围的聚合物性质。这可用于设计具有范围在数周至数月的再吸收时间的递送系统和可植入装置。Emisphere也已发表了多篇讨论肽和蛋白质的口服递送技术的文章。例如，参见Leone-bay等人的WO95/28838，其公开了由修饰的氨基酸组成的特殊载体，用来促进吸收。The VPAC2 receptor peptide agonists of the invention can be formulated for administration such that plasma levels are maintained in an effective range for extended periods of time. The major obstacles to effective oral peptide drug delivery are poor bioavailability due to acid and enzymatic degradation of the peptide, low absorption through epithelial membranes, and conversion of the peptide to insoluble after exposure to the acidic pH environment in the digestive tract form. Oral delivery systems for peptides such as those encompassed by the present invention are known in the art. For example, VPAC2 receptor peptide agonists can be encapsulated in microspheres and delivered orally. For example, VPAC2 receptor peptide agonists can be entrapped into microspheres composed of commercially available, biocompatible, biodegradable polymers, poly(lactide-co-glycolide)- COOH and olive oil were used as fillers (see Joseph et al., Diabetologia 43: 1319-1328 (2000)). Other types of microsphere technology such as Medisorb from Alkermes

and Prolease

Biodegradable polymers are also commercially available. Medisorb

Polymers can be produced with any lactide isomer. The ratio of lactide:glycolide can be varied between 0:100 and 100:0, allowing a wide range of polymer properties to be produced. This can be used to design delivery systems and implantable devices with resorption times ranging from weeks to months. Emisphere has also published several articles discussing oral delivery technologies for peptides and proteins. See, for example, WO 95/28838 to Leone-bay et al., which discloses specific carriers composed of modified amino acids to facilitate absorption.

The selective VPAC2 receptor peptide agonists described herein are useful for treating subjects with a variety of diseases and conditions. Agonists encompassed by the present invention exert their biological effects by acting on receptors, such as the VPAC2 receptor. Subjects with diseases and/or conditions that respond favorably to VPAC2 receptor stimulation or administration of VPAC2 receptor peptide agonists can thus be treated with the VPAC2 agonists of the invention. Such subjects are said to be "in need of treatment with a VPAC2 agonist" or "in need of VPAC2 receptor stimulation".

The selective VPAC2 receptor peptide agonists of the present invention are useful in the treatment of diabetes, including type 1 and type 2 diabetes (non-insulin dependent diabetes or NIDDM). Agonists can also be used to treat subjects in need of prophylactic therapy with VPAC2 receptor agonists, eg, subjects at risk of developing NIDDM. The treatment may also delay the onset of diabetes and diabetic complications. Other subjects that may be treated with the agonists of the invention include those with impaired glucose tolerance (IGT) (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Suppl. 1): S5, 1999) or subjects with abnormal fasting glucose (IFG) (Charles et al., Diabetes 40:796, 1991), those subjects who weigh more than about 25% of normal weight based on subject's height and build, have one or more Subjects whose parents have NIDDM, subjects who have had gestational diabetes, and subjects who have metabolic diseases due to, for example, decreased endogenous insulin secretion. Selective VPAC2 receptor peptide agonists can be used to prevent subjects with impaired glucose tolerance from continuing to develop NIDDM, prevent pancreatic β-cell decline, induce β-cell proliferation, improve β-cell function, activate dormant β-cells, and enable Differentiates cells into β-cells, stimulates β-cell replication, and inhibits β-cell apoptosis. Other diseases and conditions that may be treated or prevented using the agonists of the invention in the methods of the invention include: late diabetes of the young (MODY) (Herman et al., Diabetes 43:40, 1994); latent autoimmune diabetes of adults (LADA ) (Zimmet et al., Diabetes Med. 11:299, 1994); Gestational Diabetes Mellitus (Metzger, Diabetes, 40:197, 1991); Metabolic Syndrome X, Dyslipidemia, Hyperglycemia, Hyperinsulinemia, Hypertriglyceridemia Lipidemia and insulin resistance.

The selective VPAC2 receptor peptide agonists of the present invention are also useful in the treatment of secondary causes of diabetes (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Suppl. 1): S5, 1999). Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes. Drugs that can cause diabetes include, but are not limited to, medrol, niacin, corticosteroids, phenytoin, thyroxine, beta-adrenergics, alpha-interferon, and drugs used to treat HIV infection.

The selective VPAC2 receptor peptide agonists of the present invention are effective in inhibiting food intake and treating obesity.

The selective VPAC2 receptor peptide agonists of the present invention may also be effective in preventing or treating diseases such as atherosclerosis, hyperlipidemia, hypercholesterolemia, low HDL levels, Hypertension, primary pulmonary hypertension, cardiovascular disease (including atherosclerosis, coronary heart disease, and coronary artery disease), cerebrovascular disease, and peripheral vascular disease; and for the treatment of lupus, polycystic ovary syndrome, carcinogenesis (carcinogenesis), and hyperplasia, male and female reproductive problems, sexual dysfunction, ulcers, sleep disorders, lipid and carbohydrate metabolism disorders, circadian rhythm dysfunction, growth disorders, energy homeostasis disorders, immune disorders including autoimmune disorders (eg, systemic lupus erythematosus), as well as acute and chronic inflammation, rheumatoid arthritis, and septic shock.

The selective VPAC2 receptor peptide agonists of the present invention can also be used to treat physiological disorders associated with, for example, cell differentiation to produce lipid-accumulating cells, modulation of, for example, abnormal pancreatic β-cell function, pancreatic secretory tumors, and/or autoimmunity Insulin sensitivity and blood glucose levels involved in glycemia (where the hypoglycemia is due to autoantibodies to insulin, to the insulin receptor, or to stimulatory autoantibodies to pancreatic β-cells), leading to atherosclerosis Macrophage differentiation in plaques, inflammatory response, carcinogenesis, hyperplasia, adipocyte gene expression, adipocyte differentiation, decreased pancreatic β-cell mass, insulin secretion, tissue sensitivity to insulin, liposarcoma cell growth, polycystic ovary disease, persistent anovulation, hyperandrogenism, progesterone production, steroidogenesis, intracellular redox potential and oxidative stress, nitric oxide synthase (NOS) production, increased gamma-glutamyl transpeptidase, peroxidation Hydrogenase, plasma triglycerides, HDL and LDL cholesterol levels, etc.

In addition, the selective VPAC2 receptor peptide agonists of the present invention can be used in the treatment of asthma (Bolin et al., Biopolymer 37:57-66 (1995); U.S. Patent No. 5,677,419; polypeptide R3PO was shown to play a role in relaxing guinea pig tracheal smooth muscle) ; hypotension induction (VIP induces hypotension, tachycardia, and flushing in asthmatics (Morice et al., Peptides 7:279-280 (1986); Morice et al., Lancet 2:1225-1227 (1983))) ; for the treatment of male reproductive problems (Siow et al., Arch.Androl.43(1):67-71 (1999)); as an antiapoptotic/neuroprotective agent (Brenneman et al., Ann.N.Y.Acad.Sci.865 : 207-12(1998)); protect the heart in ischemic events (Kalfin et al., J.Pharmacol.Exp.Ther.1268(2):952-8(1994); Das et al., Ann.N.Y.Acad. Sci.865:297-308 (1998)); for regulation of circadian clock and related diseases (Hamar et al., Cell 109:497-508 (2002); Shen et al., Proc.Natl.Acad.Sci.97 : 11575-80, (2000)); as an antiulcer agent (Tuncel et al., Ann.N.Y.Acad.Sci.865:309-22, (1998)); and as a treatment for AIDS (Branch, et al., Blood, 106: Abstract 1427, (2005))

An "effective amount" of a selective VPAC2 receptor peptide agonist is an amount that does not cause unacceptable side effects and obtains the desired therapeutic and/or prophylactic effect when administered to a subject in need of VPAC2 receptor stimulation. A "desired therapeutic effect" includes one or more of the following: 1) amelioration of symptoms associated with the disease or disorder; 2) delay in the onset of symptoms associated with the disease or disorder; 3) increased lifespan compared to no treatment; and 4) Better quality of life compared to no treatment. For example, an "effective amount" of a VPAC2 agonist for the treatment of NIDDM is an amount that achieves greater control over blood glucose concentration and thus delays the onset of diabetic complications such as retinopathy, neuropathy or nephropathy compared to no treatment. An "effective amount" of a selective PAC2 receptor peptide agonist for the prevention of NIDDM is an amount that delays the onset of elevated blood glucose levels requiring treatment with antihypoglycemic agents such as Treatment with sulfonylureas, thiazolidinediones, insulin, and/or bisguanidines.

The "effective amount" of a selective VPAC2 receptor peptide agonist administered to a subject also depends on the type and severity of the disease, as well as the characteristics of the subject, such as general health, age, sex, body weight, and drug tolerance . The effective dose of a selective VPAC2 peptide receptor agonist to normalize blood sugar in a patient depends on a variety of factors including, but not limited to, the sex, weight and age of the subject, the severity of the failure to regulate blood sugar, the route of administration and Bioavailability, pharmacokinetic profile, potency and formulation of the peptide.

Typical dosages of the selective VPAC2 receptor peptide agonists of the invention range from about 1 μg per day to about 5000 μg per day. Preferably, the dosage ranges from about 1 μg per day to about 2500 μg per day, more preferably from about 1 μg per day to about 1000 μg per day. Even more preferably, the dosage ranges from about 5 [mu]g per day to about 100 [mu]g per day. Other preferred dosages range from about 10 [mu]g per day to about 50 [mu]g per day. Most preferably, the dosage is about 20 [mu]g per day.

A "subject" is a mammal, preferably a human, but can also be an animal such as a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.) and a laboratory animal. Room animals (eg, rats, mice, guinea pigs, etc.).

Selective VPAC2 receptor peptide agonists of the present invention can be prepared using standard methods of solid phase peptide synthesis techniques. Peptide synthesizers are commercially available, such as the Rainin-PTI Symphony Peptide Synthesizer (Tucson, AZ). Reagents for solid phase synthesis are commercially available, eg, from Glycopep (Chicago, IL). Solid-phase peptide synthesizers can be used to block interfering groups, protect amino acids to be reacted, couple, decouple, and cap unreacted amino acids according to manufacturer's instructions.

Usually, the α-N-protected amino acid and the N-terminal amino acid of the peptide chain grown on the resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidone or di Chloromethane, in the presence of coupling agents (such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole) and bases (such as diisopropylethylamine). The α-N-protecting group is removed from the resulting peptide resin using reagents such as trifluoroacetic acid or piperidine, and the coupling reaction is repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable amine protecting groups are generally known in the art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991. Examples include t-butoxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc).

Selective VPAC2 receptor peptide agonists can also be synthesized using standard automated solid phase synthesis procedures using tert-butoxycarbonyl- or fluorenylmethoxycarbonyl-α-amino acids with appropriate side chain protection. After completion of the synthesis, N-terminal modification can be accomplished by reaction of the α-amino group with, for example, (i) activated esters (using a procedure similar to that described above for the introduction of α-N-protected amino acids); (ii) aldehydes , in the presence of a reducing agent (reductive amination step); and (iii) a guanidinating agent. The peptide is then cleaved from the solid support with concurrent side chain deprotection using standard hydrogen fluoride methods or trifluoroacetic acid (TFA). The crude peptide was then further purified by reverse phase chromatography on a VYDAC C18 column using an acetonitrile gradient in 0.1% TEA. To remove acetonitrile, peptides were lyophilized from a solution containing 0.1% TEA, acetonitrile and water. Purity was verified by analytical reverse phase chromatography. Verify the identity of the peptides by mass spectrometry (Identity of peptides). Peptides are soluble in aqueous buffers at neutral pH.

The peptide agonists of the invention can also be produced by recombinant methods known in the art, using eukaryotic and prokaryotic hosts.

Once a peptide of the invention has been prepared and purified, it can be purified by covalently linking one or more PEG molecules to Cys, Lys, K(W) or K(CO(CH ₂ ) ₂ SH) residues in the peptide. Modify it. A variety of methods have been described in the art to produce peptides covalently conjugated to PEG, and the particular method used in the present invention is not meant to be limiting (for a review, see, Roberts, M. et al. Advanced Drug Delivery Reviews , 54: 459-476, 2002).

An example of a PEG molecule that can be used is Methoxy-PEG2-MAL-40K, a branched PEG maleimide (Nektar, Huntsville, Alabama). Other examples include, but are not limited to, large mPEG-SBA-20K (Nektar), mPEG2-ALD-40K (Nektar), and methoxy-PEG-MAL-30K (Dow Company (Dow)).

One method for preparing VPAC2 receptor peptide agonists involves directly linking PEG to the thiol group of the peptide using PEG-maleimide. Introduction of thiol functionality can be achieved by adding or inserting Cys or hC residues to or in the above-mentioned positions of the peptide. It is also possible to introduce a thiol functional group on the side chain of the peptide (eg, acylate the ε-amino group of lysine with a thiol-containing acid such as mercaptopropionic acid). The PEGylation process of the present invention uses Michael addition to form a stable thioether linker. The reactions are highly specific and proceed under mild conditions in the presence of other functional groups. PEG maleimide is used as a reactive polymer for the preparation of well-defined, bioactive PEG-protein conjugates. A preferred procedure uses a molar excess (preferably 1 to 10 molar excess) of the thiol-containing VPAC2 receptor peptide agonist relative to PEG maleimide to drive the reaction to completion. Preferably the reaction is carried out at room temperature between pH 4.0 and 9.0 for 10 minutes to 40 hours. The excess thiol-containing peptide not added with polyethylene glycol can be conveniently separated from the product added with polyethylene glycol by conventional separation methods. Preferably reversed phase HPLC or size exclusion chromatography is used to separate VPAC2 receptor peptide agonists. The specific conditions required for the pegylated action of VPAC2 receptor peptide agonists are presented in Example 8. Addition of cysteine to polyethylene glycol can be performed with PEG maleimide or branched PEG maleimide.

An alternative method for PEGylation of VPAC2 receptor peptide agonists involves the use of PEG-succinimide derivatives to PEGylate lysine residues. To achieve site-specific addition of polyethylene glycol, Lys residues not used for addition of polyethylene glycol can be replaced with Arg residues.

Another means of carrying out the addition of polyethylene glycol is via the Pictet-Spengler reaction. The Trp residue with its free amine is required for incorporation of PEG molecules onto VPAC2 receptor selective peptides. One means of achieving this is the site-specific introduction of a Trp residue to the amine of the side chain of Lys via an amide bond during solid phase synthesis (see Example 10).

Cyclization of VPAC2 receptor peptide agonists can be performed in solution or on a solid support. Cyclization on a solid support can be performed immediately after solid phase synthesis of the peptide. This includes selective or orthogonal protection of amino acids to be covalently linked in cyclization.

Various preferred features and embodiments of the invention will now be described with reference to the following non-limiting examples.

Example 1 - Preparation of Selective VPAC2 Receptor Peptide Agonists by Solid Phase t-Boc Chemistry:

Approximately 0.5-0.6 grams (0.38-0.45 mmol) of Boc Ser(Bzl)-PAM resin was placed in a standard 60 mL reaction vessel. Double couplings were run on an Applied Biosystems ABI430A peptide synthesizer. The following side chain protected amino acids (Boc amino acids in 2 millimolar cartridges) were obtained from Midwest Biotech (Fishers, IN) and used for synthesis:

Arg-tosyl (Tos), Asp-cyclohexyl ester (OcHx), Asp-9-fluorenylmethyl (Fm), Cys-p-methylbenzyl (p-MeBzl), Glu-cyclohexyl ester ( OcHx), His-benzyloxymethyl (Bom), Lys-2-chlorobenzyloxycarbonyl (2Cl-Z), Lys-9-fluorenylmethoxycarbonyl (Fmoc), Orn-2-chlorobenzyloxy Cylcarbonyl (2Cl-Z), Ser-O-dibenzyl ether (OBzl), Thr-O-dibenzyl ether (OBzl), Trp-formyl (CHO), Tyr-2-bromobenzyloxycarbonyl (2Br- Z), Boc-Ser (OBzl) PAM resin and MBHA resin. Trifluoroacetic acid (TFA), diisopropylethylamine (DIEA), 1.0M hydroxybenzotriazole (HOBt) in NMP, and 1.0M dicyclohexylcarbodiimide (DCC) in NMP were purchased from PE Applied Biosystems (PE-Applied Biosystems (Foster City, CA)). Dimethylformamide (DMF-Burdick and Jackson) and dichloromethane (DCM-Mallinkrodt) were purchased from Mays Chemical Co. (Indianapolis, IN). Benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) was obtained from Nova Biochem (San Diego, CA).

Standard double couplings were performed using either symmetrical anhydrides or HOBt esters, both formed with DCC. At the completion of the synthesis, the N-terminal Boc group was removed and, if Trp was present in the sequence, the peptidyl resin was treated with 20% piperidine in DMF to deformylate the Trp side chain. For acylation of the N-terminus, a 4-fold excess of the symmetrical anhydride of the corresponding acid was added to the peptide resin. The symmetrical anhydrides were prepared by activation with diisopropylcarbodiimide (DIC) in DCM. The reaction was carried out for 4 hours and monitored by ninhydrin detection. After washing with DCM, the resin was transferred to a TEFLON reaction vessel and dried in vacuo.

Cutting was performed by contacting the reaction vessel with a HF (hydrofluoric acid) unit (Penninsula Laboratories). Add 1 mL m-cresol per gram/resin and concentrate into 10 mL HF (purchased from AGA Corporation, Indianapolis, IN) in a pre-cooled vessel. When methionine is present, then add 1 mL of DMS per gram of resin. The reaction was stirred for 1 hour in an ice bath. HF was removed in vacuo. The residue was suspended in ether. The solid was filtered and washed with ether. Each peptide was extracted into aqueous acetic acid and lyophilized or loaded directly onto a reversed-phase column.

Purification was performed on a 2.2 x 25 cm VYDAC C18 column in buffer A (0.1% TFA in water). A gradient of 20% to 90% B (0.1% TFA in acetonitrile) was run at 10 mL/min over 120 min on HPLC (Waters) while UV was monitored at 280 nm (4.0 A) and collected for one minute fraction. Appropriate fractions were pooled, frozen and lyophilized. The dried product was analyzed by HPLC (0.46 x 15 cm METASIL AQ C18) and MALDI mass spectrometry.

Cyclic rings with lactam bridges linking lysine and aspartate residues were prepared by selectively protecting the side chains of lysine and aspartate residues with Fmoc and Fm, respectively. VPAC2 receptor peptide agonist. All other amino acids used in the synthesis were standard, benzyl side chain protected Boc-amino acids. Following solid phase synthesis of the peptide, cyclization on the solid support can be performed immediately again. The Fmoc and Fm protecting groups were selectively removed and cyclization was performed by activating the carboxyl group of aspartic acid with BOP in the presence of DIEA. The reaction was allowed to proceed for 24 hours, monitored by ninhydrin detection.

Example 2 - Preparation of Selective VPAC2 Receptor Peptide Agonists by Solid Phase Fmoc Chemistry:

Approximately 114 mg (50 mmol) of FMOC Ser(tBu) WANG resin (available from Glycopep, Chicago, IL) was added to each reaction vessel. Synthesis was performed on a RaininSymphony peptide synthesizer. Analogs with a C-terminal amide were prepared with 75 mg (50 micromolar) of Rink Amide AM resin (Rapp Polyme re. Tuebingen, Germany).

The following Fmoc amino acids were purchased from Glycopep (Chicago, IL) and Novartis Biochem (La Jolla, CA): Arg-2,2,4,6,7-5 Methyldihydrobenzofuran-5-sulfonyl (Pbf), Asn-trityl (Trt), Asp-β-tert-butyl ester (tBu), Asp-β-allyl ester (Allyl), Glu-δ-tert-butyl ester (tBu), Glu-δ-allyl ester (Allyl), Gln-trityl (Trt), His-trityl (Trt), Lys-tert-butoxycarbonyl (Boc), Lys-allyloxycarbonyl (Aloc), Orn-allyloxycarbonyl (Aloc), Ser-tert-butyl ether (OtBu), Thr-tert-butyl ether (OtBu), Trp-tert-butyl Oxycarbonyl (Boc), Tyr-tert-butyl ether (OtBu).

Solvents dimethylformamide (DMF-Burdick and Jackson), N-methylpyrrolidone (NMP-Burdick and Jackson), dichloromethane (DCM-Mallinkrodt) were purchased from Mays Chemical Co. (Indianapolis, IN).

Hydroxybenzotriazole (HOBt), diisopropylcarbodiimide (DIC), diisopropylethylamine (DIEA) and piperidine (Pip) were purchased from Aldrich Chemical Co (Milwaukee , WI). Benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) was obtained from NovaBiochem (San Diego, CA).

All amino acids were dissolved in DMF at 0.3M. After deprotection with 20% piperidine/DMF for 20 minutes, a DIC/HOBt activated coupling was performed for three hours. Each resin was washed with DMF after deprotection and coupling. After the final coupling and deprotection, the peptidyl resin was washed with DCM and dried in a vacuum reaction vessel. For N-terminal acylation, a 4-fold excess of the symmetrical anhydride of the corresponding acid was added to the peptide resin. Symmetrical anhydrides were prepared by DIC activation in DCM. The reaction was carried out for 4 hours and monitored with ninhydrin detection. The peptide resin was then washed with DCM and dried in vacuo.

The cleavage reaction was mixed for 2 h with a cleavage mixture consisting of 0.2 mL of thiophenylmethane, 0.2 mL of methanol, and 0.4 mL of triisopropylsilane per 10 mL of TFA, all purchased from Aldrich Chemical Co., Milwaukee, WI). If Cys is present in the sequence, add 2% ethanedithiol. To the TFA filtrate was added 40 mL of diethyl ether. The precipitant was centrifuged at 2000 rpm for 2 minutes. Pour off the supernatant. The pellet was resuspended in 40 mL of diethyl ether, centrifuged again, decanted, dried under nitrogen, and then dried in vacuo.

0.3-0.6 mg of each product was dissolved in 1 ml of 0.1% TFA/acetonitrile (ACN), of which 20 μL was analyzed by HPLC [0.46×15 cm METASIL AQ C18, 1 mL/min, 45 ° C, 214 nM (0.2 A), A = 0.1% TFA, B = 0.1% TFA/50% ACN. Gradient = 50% B to 90% B over 30 minutes].

Purification was performed on a 2.2 x 25 cm VYDAC C18 column in buffer A (0.1% TFA in water). A gradient of 20% to 90% B (0.1% TFA in acetonitrile) was run at 10 mL/min on HPLC (Waters) over 120 minutes while monitoring UV at 280 nm (4.0 A) and collecting a minute fraction. Appropriate fractions were pooled, frozen and lyophilized. The dried product was analyzed by HPLC (0.46 x 15 cm METASIL AQ C18) and MALDI mass spectrometry.

Cyclic rings with lactam bridges linking lysine and aspartate residues were prepared by selectively protecting the side chains of lysine and aspartate residues with Aloc and Allyl, respectively. VPAC2 receptor peptide agonist. All other amino acids used in the synthesis were standard, tert-butyl side chain protected Fmoc-amino acids.

Following solid phase synthesis of the peptide, cyclization on the solid support can be performed immediately again. The Aloc and Allyl protecting groups were selectively removed and cyclization was performed by activating the carboxyl group of aspartic acid with BOP in the presence of DIEA.

Preparation of P603 by solid-phase Fmoc chemistry

Approximately 75 mg (50 micromoles) of polystyrene Rink Amide AM resin (Rapp Polymere GmbH, Tubingen, Germany) was placed in the reaction vessel. Fmoc-Lys-allyloxycarbonyl (Aloc) was used in the first synthesis cycle of automated synthesis using a Rainin Symphony peptide synthesizer. Extension to the peptide resin was performed as described in Example 2 above. After completion of the automatic extension of the peptide resin (including C6-N-terminal acylation), using in DCM-acetic acid-piperidine (92:5:3, v/v/v) (Aldrich Chemical Co., Milwaukee, WI) Tetrakis (triphenylphosphine) palladium (0) [100 micromole] was used to manually remove the Aloc protecting group, which was carried out at 25°C for 20 minutes. This step was repeated twice. The aloc was then washed with 5% DIEA in DCM and 0.03M sodium diethyldithiocarbamate trihydrate (Aldrich Chemical Co., Milwaukee, WI) in DMF. protective resin. Fmoc-Glu-α-OtBu ester (500 micromolar; purchased from NovaBiochem, La Jolla, CA) was artificially incorporated using DIC (500 micromolar) and HOBt (500 micromolar) in DMF. ), which was carried out at 25°C for 2 hours. After subsequent Fmoc removal, palmitic acid (500 micromolar; purchased from Aldrich Chemical Co., Milwaukee, WI) was incorporated using the same method as that used for the Fmoc-Glu-α-OtBu ester. Cleavage of the peptide from the resin and purification was performed as described in Example 2.

Example 3 - In vitro potency at the human VPAC2 receptor

α screening: cells (CHO-S cells stably expressing human VPAC2 receptor) were washed once with PBS in a culture flask. Cells were then rinsed with enzyme-free dissociation buffer. Remove dissociated cells. Cells were then spun down and washed in stimulation buffer. For each data point, 50,000 cells suspended in stimulation buffer were used. To this buffer, alpha selection receptor beads are added along with stimuli. The mixture was incubated for 60 minutes. Add Lysis Buffer and Alpha Screen Donor Beads and incubate for 60 to 120 minutes. The alpha screen signal (indicative of intracellular cAMP levels) is read on a suitable instrument (eg, AlphaQuest from Perkin-Elmer). The steps involving alpha screening of donor and acceptor beads were performed under low light. _EC50 for cAMP production was calculated from the raw signal or based on absolute cAMP levels determined using a standard curve performed on each plate. Results for each agonist are from at least two analyzes performed in a single run. For some agonists, results are the average of more than one run. The peptide concentrations tested were: 10000, 1000, 100, 10, 3, 1, 0.1, 0.01, 0.003, 0.001, 0.0001 and 0.00001 nM.

DiscoverRx: The day before the assay, a CHO-S cell line stably expressing the human VPAC2 receptor was seeded in 96-well microtiter plates at 50,000 cells/well. Cells were allowed to attach for 24 hours in 200 μL of medium. On the day of the experiment, the medium was removed. And wash the cells twice. Cells were incubated in assay buffer plus IBMX for 15 minutes at room temperature. Then, stimuli were added and dissolved in assay buffer. The stimuli were present for 30 minutes. Then, gently remove the assay buffer. Add the Cell Lysis Reagent from the DiscoverRx cAMP Kit. Then, the standard protocol for visualization of cAMP signal described by the manufacturer (DiscoveRx Inc., USA) was used. _EC50 values for cAMP generation were calculated from the raw signal or based on absolute cAMP levels determined using a standard curve performed on each plate. Typical assay concentrations for peptides are: 1000, 300, 100, 10, 1, 0.3, 0.1, 0.01, 0.001, 0.0001 and OnM.

Table 1 reports the activity ( _EC50 (nM)) of the human VPAC2 receptor in different assay formats.

Table 1. Peptide titers at the human VPAC2 receptor

Example 4 - Selectivity

Binding assays: Membranes prepared from the stable VPAC2 cell line (see Example 3) or from cells transiently transfected with human VPAC1 or PAC1 were used. Filter binding assays were performed using 125I-labeled PACAP-27 as a tracer for VPAC1 and VPAC2, as well as PAC1.

Solutions and instruments used for this assay include:

Presoak: 0.5% polyvinylamine in distilled water

Buffer for washing filter plates: 25mM HEPES pH7.4

Blocking buffer: 25mM HEPES pH7.4; 0.2% protease-free BSA

Assay buffer: 25mM HEPES pH7.4; 0.5% protease-free BSA

Dilution and assay plate: PS-Microplate, U-shaped

Filter plate: Multiscreen FB Opaque plate; 1.0μM B-type Glasfiber filter membrane

To prepare the filter plates, the presoak was aspirated by vacuum filtration. Plates were washed twice with 200 μL wash buffer. Add 200 µL of blocking buffer to the filter plate. Filter plates were then incubated with 200 μL of presoak solution for 1 hour at room temperature.

Load the assay plate with 25 μL of assay buffer, 25 μL (2.5 μg) of membrane suspended in assay buffer, 25 μL of compound (agonist) in assay buffer, and 25 μL of tracer in assay buffer ( about 40000cpm). The mounted plates were incubated with shaking for 1 hour.

Perform transfer from assay plate to filter plate. Blocking buffer was aspirated by vacuum filtration and washed twice with wash buffer. Transfer 90 μL from the assay plate to the filter plate. Aspirate the 90 μL transferred from the assay plate and wash three times with 200 μL wash buffer. Remove the plastic support. Dry at 60°C for 1 hour. Add 30 μL Microscint. count.

Example 5 - In Vitro Potency of Rat VPAC1 and VPAC2 Receptors:

DiscoveRx: CHO-PO cells were transiently transfected with rat VPAC1 or VPAC2 receptor DNA using a commercially available transfection reagent (Lipofectamine from Invitrogen). Cells were seeded in 96-well plates at a density of 10,000/well and allowed to grow for 3 days in 200 mL of medium. On day 3, assays were performed.

On the day of the experiment, the medium was removed. And wash the cells twice. Cells were incubated in assay buffer plus IBMX for 15 minutes at room temperature. Then, stimuli were added and dissolved in assay buffer. The stimuli were present for 30 minutes. Then, gently remove the assay buffer. Add the Cell Lysis Reagent from the DiscoverRx cAMP Kit. Then, the standard protocol for visualization of cAMP signal described by the manufacturer (DiscoveRx Inc., USA) was used. _EC50 values for cAMP generation were calculated from the raw signal or based on absolute cAMP levels determined using a standard curve performed on each plate. Typical assay concentrations for peptides are: 1000, 300, 100, 10, 1, 0.3, 0.1, 0.01, 0.001, 0.0001 and OnM.

Example 6 - In vivo assay:

Intravenous glucose tolerance test (IVGTT): ordinary Wistar rats were fasted overnight and anesthetized before the experiment. Insert the blood sampling catheter into the rat. The agonist is usually given subcutaneously 24 hours before the glucose load. Blood samples were taken from the carotid artery. Blood samples were taken immediately before agonist and glucose injections. Following the initial blood sample, mixed dextrose was injected intravenously (i.v.). A glucose load of 0.5 g/kg body weight was given, with a total of 1.5 mL of vehicle with glucose and agonist injected per kg body weight. Peptide concentrations were varied in μg/kg to yield the required doses. Blood samples were taken 2, 4, 6 and 10 minutes after glucose administration. A control group of animals received the same vehicle with only glucose but no agonist added. In some instances, 20 and 30 minute post-glucose blood samples were taken. Add aprotinin (250-500kIU/ml blood) to the blood sample. Plasma was then analyzed for glucose and insulin by standard methods.

Prepared and calibrated peptides stored in PBS were used. Typically, this stock solution is a pre-diluted 100 [mu]M stock solution. However, a more concentrated stock solution with about 1 mg of agonist per mL was used. The specific concentration is generally known. Variability in maximal response was mostly due to variability in vehicle dose. The protocol details are as follows:

Species/Strain/Bodyweight Rat/Wistar Unilever/about 275-300g treatment schedule single dose Dosing Volume/Route 1.5mL/kg/intravenous (iv) carrier 8% PEG300, 0.1% BSA in water Food/Water Program Rats were fasted overnight before surgery. Life State Parameters Animals were sacrificed at the end of the test. IVGTT: performed on each group of rats (with two catheters, jugular vein and carotid artery), anesthetized with pentobarbital. Dextrose IV bolus: 500 mg/kg as a 10% solution (5 mL/kg) at time=0. Compounds intravenously (iv): 0-240 min before glucose blood sampling (300 μL from carotid artery; EDTA as anticoagulant; aprotinin and PMSF as antiproteolysates; on ice): 0, 2, 4, 6 and 10, 20 and 30 minutes. Determined parameters: insulin + glucose Toxicokinetics Plasma samples remaining after insulin measurements were stored at -20°C and compound levels were determined.

Embodiment 7-Stability study of rat serum:

In order to determine the stability of VPAC2 receptor peptide agonists in rat serum, CHO-VPAC2 cell clone #6 (96-well plate/50,000 cells/well, cultured for 1 day), PBS1×(Gibco), in 100 μM stock was obtained. Peptides for analysis in solution, rat serum from sacrificed normal Wistar rats, aprotinin and DiscoverRx assay kit. Rat sera were stored at 4 °C prior to use and used within two weeks.

On day 0, two 100 μL aliquots of 10 μM peptide in rat serum were prepared by adding 10 μL of the peptide stock to 90 μL rat serum for each aliquot. Add 250 kIU aprotinin/mL to one of these aliquots. Aliquots were stored at 4°C in the presence of aprotinin. Aliquots were stored at 37°C in the absence of aprotinin. Aliquots were incubated for 24 hours.

On day 1, after 24 hours incubation of the aliquot prepared on day 0, an incubation buffer was prepared containing PBS + 1.3 mM _CaCl2 , 1.2 mM MgCl2, ₂ mM glucose and 0.5 mM IBMX. For each peptide studied, plates were prepared with 11 serial 3x dilutions of the peptide in serum (for 4°C and 37°C aliquots). The maximum concentration used was 4000 nM. Plates containing cells were washed twice in incubation buffer and cells were incubated for 15 minutes with 50 μL of incubation medium per well. Transfer 50 μL per well of the solution to the cells, using the maximum concentration suggested by the primary screen, in duplicate, from the plate prepared with 11 aliquots at 4°C and 37°C for each peptide studied Serial 3x diluted peptides. This step dilutes the peptide concentration by a factor of 2. Cells were incubated at room temperature for 30 minutes. Remove the supernatant. Add 40 μL/well of DiscoverRx antibody/extraction buffer. Cells were incubated on a shaker (300 rpm) for 1 hour. The usual procedure for the DiscoverRx kit was then followed. In column 12 a cAMP standard is included. _EC50 values were determined from cAMP assay data. The remaining amount of active peptide for each condition was estimated by the formula EC50 _{, 4°C} /EC50 _{, 37°C} .

Table 5 Predicted Peptide Stability After 24 Hours in Rat Serum at 37°C

Agonist# % Stability ¹ P603 67

¹ A value >100% may represent intact peptide released from PEG conjugate

Example 8 - Selective VPAC2 receptor peptide agonists using thiol-based chemistry Effect of adding polyethylene glycol:

The addition of polyethylene glycol is generally carried out under conditions that allow the formation of thioether linkages. In particular, the pH of the solution ranges from about 4 to 9, and the concentration of the thiol-containing peptide ranges from 0.7 to 10 molar excess of the PEG maleimide concentration. The polyethylene glycol addition reaction is generally carried out at room temperature. The VPAC2 receptor peptide agonists were then isolated by reverse phase HPLC or size exclusion chromatography (SEC). Peptide analogs spiked with polyethylene glycol were characterized by analytical RP-HPLC, HPLC-SEC, SDS-PAGE and/or MALDI mass spectrometry.

Sulfur is generally added by adding cysteine or homocysteine or a thiol-containing moiety at one or both ends, or by inserting a cysteine or homocysteine or a thiol-containing moiety into the sequence. Hydroxyl functional groups are introduced into or onto selective VPAC2 receptor peptide agonists. A thiol-containing VPAC2 receptor peptide agonist is reacted with 40 kDa, 30 kDa, or 20 kDa PEG maleimide to produce derivatives covalently linked to PEG via a thioether bond.

Example 9 - Addition of polyethylene glycol via acylated lysine side chains

To achieve site-specific PEGylation of selective VPAC2 receptor peptide agonists, all Lys residues except those for which PEGylation was planned were converted to Arg residues. A PEG molecule that can be used is mPEG-SBA-20K (Nektar, Lot: PT-04E-11). Preferably, the polyethylene glycol addition reaction is carried out at room temperature for 2-3 hours. Peptides were purified by preparative HPLC.

Example 10 - Addition of polyethylene glycol via Pictet-SDengler reaction

For PEG addition via the Pictet-Spengler reaction, a Trp residue with its free amine is required to incorporate the PEG molecule onto a selective VPAC2 receptor peptide agonist. One way to achieve this is to couple the Trp residue to the side chain of Lys. A deep SAR indicates that the modification did not alter the properties of the parental peptide in terms of its in vitro potency and selectivity.

The reaction uses PEG with functional aldehydes, such as mPEG2-BUTYRALD-40K (Nektar, USA). The site-specific addition of polyethylene glycol involves the formation of a tetracarboline ring between the PEG and the peptide. The addition of polyethylene glycol is carried out in glacial acetic acid at room temperature for 1 to 48 hours. The reaction uses a 1 to 10 molar excess of PEG aldehyde. After removal of acetic acid, VPAC2 receptor peptide agonists were isolated by preparative RP-HPLC.

Other modifications of the invention will be apparent to those skilled in the art without departing from the scope of the invention.

sequence listing

<110> Eli Lilly Company

Zhang Lianshan

J Alsina-Fernandez

<120> Selective VPAC2 receptor peptide agonist

<130>X17364 WO

<150>US 60/743,366

<151>2006-02-28

<160>8

<170> PatentIn version 3.3

<210>1

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: VPAC2 receptor peptide agonist

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>Xaa may or may not be present. If present, Xaa is His or dH

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> Xaa is dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa is Asp or Glu

<220>

<221>MISC_FEATURE

<222>(4)..(4)

<223>Xaa is Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or NMeA

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223>Xaa is Val, Leu, Phe, Ile, Thr, Trp, Tyr, dV, Aib, or NMeV

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223>Xaa is Phe, Ile, Leu, Thr, Val, Trp, or Tyr

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223>Xaa is Asp, Glu, Ala, Lys, Leu, Arg, or Tyr

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223>Xaa is Asn, Gln, Glu, Ser, Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(10)..(10)

<223>Xaa is Tyr, Trp, or Tyr(OMe)

<220>

<221>MISC_FEATURE

<222>(12)..(12)

<223>Xaa is Arg, Lys, hR, Orn, Aib, Ala, Leu, Gln, Phe, or Cys

<220>

<221>MISC_FEATURE

<222>(13)..(13)

<223>Xaa is Leu, Phe, Glu, Ala, Aib, Ser, Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(14)..(14)

<223> Xaa is Arg, Leu, Lys, Ala, hR, Orn, Phe, Gln, Aib, or Cit

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> Xaa is Lys, Ala, Arg, Glu, Leu, Orn, Phe, Gln, Aib, K(Ac), Cys, K(W), or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223>Xaa is Gln, Lys, Ala, Ser, Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223>Xaa is Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(18)..(18)

<223> Xaa is Ala, Ser, Cys, or Abu

<220>

<221>MISC_FEATURE

<222>(19)..(19)

<223>Xaa is Ala, Leu, Gly, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or Abu

<220>

<221>MISC_FEATURE

<222>(20)..(20)

<223> Xaa is Lys, Gln, hR, Arg, Ser, Orn, Ala, Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac),

Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(21)..(21)

<223>Xaa is Lys, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Ser, Cys, K(W), K(CO(CH ₂ ) ₂ SH),

or hC

<220>

<221>MISC_FEATURE

<222>(22)..(22)

<223>Xaa is Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib, or Ser

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> Xaa is Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib, or Ser

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223>Xaa is Gln, Asn, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(25)..(25)

<223>Xaa is Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr, Aib, Glu, Cys, K(CO(CH ₂ ) ₂ SH), or hC

<220>

<221>MISC_FEATURE

<222>(26)..(26)

<223>Xaa is Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(27)..(27)

<223>Xaa is Lys, hR, Arg, Gln, Orn, or dK

<220>

<221>MISC_FEATURE

<222>(28)..(28)

<223>Xaa is Asn, Gln, Lys, Arg, Aib, Orn, hR, Pro, dK, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(29)..(32)

<223> Any Xaa may or may not be present. If any Xaa is absent, the next amino acid present downstream is in the peptide agonist sequence

the next amino acid of

<220>

<221>MISC_FEATURE

<222>(29)..(29)

<223>Xaa may or may not be present. If present, Xaa is Lys, Ser, Arg, Asn, hR, Cys, or Orn

<220>

<221>MISC_FEATURE

<222>(30)..(30)

<223>Xaa may or may not be present. If present, Xaa is Arg, Lys, Ile, or hR

<220>

<221>MISC_FEATURE

<222>(31)..(31)

<223>Xaa may or may not be present. If present, Xaa is Tyr, His, Phe, or Gln

<220>

<221>MISC_FEATURE

<222>(32)..(32)

<223>Xaa may or may not be present. If present, Xaa is Cys

<400>1

<210>2

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: VPAC2 receptor peptide agonist

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223> Xaa is Asp or Glu

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223>Xaa is Asp, Glu, Ala, Lys, Leu, Arg, or Tyr

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223>Xaa is Asn, Gln, Glu, Ser, Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(10)..(10)

<223>Xaa is Tyr, Trp, or Tyr(OMe)

<220>

<221>MISC_FEATURE

<222>(12)..(12)

<223>Xaa is Arg, Lys, hR, Orn, Aib, Ala, Leu, Gln, Phe, or Cys

<220>

<221>MISC_FEATURE

<222>(13)..(13)

<223>Xaa is Leu, Phe, Glu, Ala, Aib, Ser, Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(14)..(14)

<223> Xaa is Arg, Leu, Lys, Ala, hR, Orn, Phe, Gln, Aib, or Cit

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> Xaa is Lys, Ala, Arg, Glu, Leu, Orn, Phe, Gln, Aib, K(Ac), Cys, K(W), or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223>Xaa is Gln, Lys, Ala, Ser, Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223>Xaa is Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(18)..(18)

<223> Xaa is Ala, Ser, Cys, or Abu

<220>

<221>MISC_FEATURE

<222>(19)..(19)

<223>Xaa is Ala, Leu, Gly, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or Abu

<220>

<221>MISC_FEATURE

<222>(20)..(20)

<223> Xaa is Lys, Gln, hR, Arg, Ser, Orn, Ala, Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cys, or K(CO(CH ₂ ) ₂ SH)

<220>

<221>MISC_FEATURE

<222>(21)..(21)

<223>Xaa is Lys, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Ser, Cys, K(W), K(CO(CH ₂ ) ₂ SH), or hC

<220>

<221>MISC_FEATURE

<222>(22)..(22)

<223>Xaa is Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib, or Ser

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> Xaa is Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib, or Ser

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223>Xaa is Gln, Asn, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(25)..(25)

<223>Xaa is Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr, Aib, Glu, Cys, K(CO(CH ₂ ) ₂ SH), or hC

<220>

<221>MISC_FEATURE

<222>(26)..(26)

<223>Xaa is Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(27)..(27)

<223> Xaa is Lys, hR, Arg, Gln, Orn, or dK

<220>

<221>MISC_FEATURE

<222>(28)..(28)

<223>Xaa is Asn, Gln, Lys, Arg, Aib, Orn, hR, Pro, dK, Cys, K(CO(CH ₂ ) ₂ SH), or K(W)

<220>

<221>MISC_FEATURE

<222>(29)..(32)

<223> Any Xaa may or may not be present. If any Xaa is absent, the next amino acid present downstream is the next amino acid in the peptide agonist sequence

<220>

<221>MISC_FEATURE

<222>(29)..(29)

<223>Xaa may or may not be present. If present, Xaa is Lys, Ser, Arg, Asn, hR, Cys, or Orn

<220>

<221>MISC_FEATURE

<222>(30)..(30)

<223>Xaa may or may not be present. If present, Xaa is Arg, Lys, Ile, or hR

<220>

<221>MISC_FEATURE

<222>(31)..(31)

<223>Xaa may or may not be present. If present, Xaa is Tyr, His, Phe, or Gln

<220>

<221>MISC_FEATURE

<222>(32)..(32)

<223>Xaa may or may not be present. If present, Xaa is Cys

<400>2

<210>3

<211>28

<212>PRT

<213> Human (Homo sapiens)

<400>3

<210>4

<211>27

<212>PRT

<213> people

<220>

<221>MOD_RES

<222>(27)..(27)

<223> amidation

<400>4

<210>5

<211>37

<212>PRT

<213> people

<220>

<221>MOD_RES

<222>(37)..(37)

<223> amidation

<400>5

<210>6

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: N-terminal modification of VPAC2 receptor agonist, where Arg at position 9 is linked to the N-terminus of the peptide agonist

<400>6

<210>7

<211>39

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: VPAC2 receptor peptide agonist P603

<220>

<221>MOD_RES

<222>(1)..(1)

<223>C6 acylation

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Methoxy

<220>

<221>MOD_RES

<222>(12)..(12)

<223>Orn

<220>

<221>MOD_RES

<222>(15)..(15)

<223> Aib

<220>

<221>MOD_RES

<222>(19)..(19)

<223>Abu

<220>

<221>MOD_RES

<222>(20)..(20)

<223> Aib

<220>

<221>MOD_RES

<222>(21)..(21)

<223>Orn

<220>

<221>MOD_RES

<222>(23)..(23)

<223> Aib

<220>

<221>MOD_RES

<222>(25)..(25)

<223> Aib

<220>

<221>MOD_RES

<222>(27)..(28)

<223>Orn

<220>

<221>MOD_RES

<222>(39)..(39)

<223> The C-terminal lysine residue has attached to its side chain a glutamic acid residue acylated with palmitic acid at the α-amino group

<220>

<221>MOD_RES

<222>(39)..(39)

<223> amidation

<400>7

<210>8

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: C-terminal extension of VPAC2 receptor peptide agonist

<220>

<223>C-terminal amino acid can be amidated

<220>

<221>MOD_RES

<222>(11)..(11)

<223> The C-terminal lysine residue has attached to its side chain a glutamic acid residue acylated with palmitic acid at the α-amino group

<400>8

Claims (23)

1. VPAC2 receptor peptide agonist, it comprises the sequence of following formula: Xaa _{1 -Xaa 2} -Xaa ₃ -Xaa ₄ -Xaa ₅ -Xaa ₆ -Thr-Xaa 8 -Xaa ₉ -Xaa 10 -Thr-Xaa _{12 -Xaa 13} -Xaa ₁₄ -Xaa ₁₅ -Xaa ₁₆ -Xaa ₁₇ -Xaa ₁₈ -Xaa ₁₉ -Xaa ₂₀ -Xaa ₂₁ -Xaa ₂₂ -Xaa ₂₃ -Xaa ₂₄ -Xaa ₂₅ -Xaa 26 -Xaa ₂₇ -Xaa ₂₈ -Xaa _{29 -Xaa 30} -Xaa ₃₁ -Xaa ₃₂ Formula 1 (SEQ ID NO: 1) in: Xaa ₁ is His, dH or absent; Xaa ₂ is dA, Ser, Val, Gly, Thr, Leu, dS, Pro or Aib; Xaa ₃ is Asp or Glu; Xaa ₄ is Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib or NMeA; Xaa ₅ is Val, Leu, Phe, Ile, Thr, Trp, Tyr, dV, Aib or NMeV; Xaa ₆ is Phe, Ile, Leu, Thr, Va1, Trp or Tyr; Xaa ₈ is Asp, Glu, Ala, Lys, Leu, Arg or Tyr; Xaa ₉ is Asn, Gln, Glu, Ser, Cys or K(CO(CH ₂ ) ₂ SH); Xaa ₁₀ is Tyr, Trp or Tyr(OMe); Xaa ₁₂ is Arg, Lys, hR, Orn, Aib, Ala, Leu, Gln, Phe or Cys; Xaa ₁₃ is Leu, Phe, Glu, Ala, Aib, Ser, Cys or K(CO(CH ₂ ) ₂ SH); Xaa ₁₄ is Arg, Leu, Lys, Ala, hR, Orn, Phe, Gln, Aib or Cit; Xaa ₁₅ is Lys, Ala, Arg, Glu, Leu, Orn, Phe, Gln, Aib, K(Ac), Cys, K(W) or K(CO(CH ₂ ) ₂ SH); Xaa ₁₆ is Gln, Lys, Ala, Ser, Cys or K(CO(CH ₂ ) ₂ SH); Xaa ₁₇ is Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W); Xaa ₁₈ is Ala, Ser, Cys or Abu; Xaa ₁₉ is Ala, Leu, Gly, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or Abu; Xaa ₂₀ is Lys, Gln, hR, Arg, Ser, Orn, Ala, Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cys or K(CO( _CH2 ) _2SH ) ; Xaa ₂₁ is Lys, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Ser, Cys, K(W), K(CO( _CH2 ) _2SH ) or hC; Xaa ₂₂ is Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib or Ser; Xaa ₂₃ is Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib or Ser; Xaa ₂₄ is Gln, Asn, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or K(W); Xaa ₂₅ is Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr, Aib, Glu, Cys, K(CO( _CH2 ) _2SH ) or hC; Xaa ₂₆ is Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W); Xaa ₂₇ is Lys, hR, Arg, Gln, Orn or dK; Xaa ₂₈ is Asn, Gln, Lys, Arg, Aib, Orn, hR, Pro, dK, Cys, K(CO( _CH2 ) _2SH ) or K(W); Xaa ₂₉ is Lys, Ser, Arg, Asn, hR, Cys, Orn or absent; Xaa ₃₀ is Arg, Lys, Ile, hR or absent; Xaa ₃₁ is Tyr, His, Phe, Gln or absent; and Xaa ₃₂ is Cys or absent; and satisfies that if Xaa ₂₉ , Xaa ₃₀ , Xaa ₃₁ or Xaa ₃₂ are absent, the next amino acid present downstream is the next amino acid in the sequence of the peptide agonist; And, the agonist comprises a C-terminal extension comprising the following amino acid sequence: GGPSSGAPPPK (EC ₁₆ ) Wherein, the C-terminal amino acid may be amidated. 2. The VPAC2 receptor peptide agonist according to claim 1, comprising a sequence of the formula: His-Ser-Xaa ₃ -Ala-Val-Phe-Thr-Xaa ₈ -Xaa ₉ -Xaa ₁₀ -Thr-Xaa ₁₂ -Xaa ₁₃ -Xaa ₁₄ -Xaa ₁₅ -Xaa 16 -Xaa ₁₇ -Xaa _{18 -Xaa 19} - Xaa ₂₀ -Xaa ₂₁ -Xaa ₂₂ -Xaa ₂₃ -Xaa ₂₄ -Xaa ₂₅ -Xaa ₂₆ -Xaa ₂₇ -Xaa ₂₈ -Xaa ₂₉ -Xaa ₃₀ -Xaa ₃₁ -Xaa ₃₂ Formula 2 (SEQ ID NO: 2) in: Xaa ₃ is Asp or Glu; Xaa ₈ is Asp, Glu, Ala, Lys, Leu, Arg or Tyr; Xaa ₉ is Asn, Gln, Glu, Ser, Cys or K(CO(CH ₂ ) ₂ SH); Xaa ₁₀ is Tyr, Trp or Tyr(OMe); Xaa ₁₂ is Arg, Lys, hR, Orn, Aib, Ala, Leu, Gln, Phe or Cys; Xaa ₁₃ is Leu, Phe, Glu, Ala, Aib, Ser, Cys or K(CO(CH ₂ ) ₂ SH); Xaa ₁₄ is Arg, Leu, Lys, Ala, hR, Orn, Phe, Gln, Aib or Cit; Xaa ₁₅ is Lys, Ala, Arg, Glu, Leu, Orn, Phe, Gln, Aib, K(Ac), Cys, K(W) or K(CO(CH ₂ ) ₂ SH); Xaa ₁₆ is Gln, Lys, Ala, Ser, Cys or K(CO(CH ₂ ) ₂ SH); Xaa ₁₇ is Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W); Xaa ₁₈ is Ala, Ser, Cys or Abu; Xaa ₁₉ is Ala, Leu, Gly, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or Abu; Xaa ₂₀ is Lys, Gln, hR, Arg, Ser, Orn, Ala, Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cys or K(CO( _CH2 ) _2SH ) ; Xaa ₂₁ is Lys, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Ser, Cys, K(W), K(CO( _CH2 ) _2SH ) or hC; Xaa ₂₂ is Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib or Ser; Xaa ₂₃ is Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib or Ser; Xaa ₂₄ is Gln, Asn, Ser, Cys, K(CO(CH ₂ ) ₂ SH) or K(W); Xaa ₂₅ is Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr, Aib, Glu, Cys, K(CO( _CH2 ) _2SH ) or hC; Xaa ₂₆ is Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, K(CO( _CH2 ) _2SH ) or K(W); Xaa ₂₇ is Lys, hR, Arg, Gln, Orn or dK; Xaa ₂₈ is Asn, Gln, Lys, Arg, Aib, Orn, hR, Pro, dK, Cys, K(CO( _CH2 ) _2SH ) or K(W); Xaa ₂₉ is Lys, Ser, Arg, Asn, hR, Cys, Orn or absent; Xaa ₃₀ is Arg, Lys, Ile, hR or absent; Xaa ₃₁ is Tyr, His, Phe, Gln or absent; and Xaa ₃₂ is Cys or absent; and satisfies that if Xaa ₂₉ , Xaa ₃₀ , Xaa ₃₁ or Xaa ₃₂ are absent, the next amino acid present downstream is the next amino acid in the sequence of the peptide agonist; And, the agonist comprises a C-terminal extension comprising the following amino acid sequence: GGPSSGAPPPK (EC ₁₆ ) Wherein, the C-terminal amino acid may be amidated. 3. A VPAC2 receptor peptide agonist according to any one of the preceding claims, wherein Xaa ₈ is Glu, Xaa ₉ is GIn, and Xaa ₁₀ is Tyr(OMe). 4. A VPAC2 receptor peptide agonist according to any one of the preceding claims, wherein Xaa ₁₅ is Aib. 5. A VPAC2 receptor peptide agonist according to any one of the preceding claims, wherein Xaa ₂₀ is Aib. 6. A VPAC2 receptor peptide agonist according to any one of the preceding claims, wherein _Xaa12 , _Xaa21 , _Xaa27 and _Xaa28 are all Orn. 7. A VPAC2 receptor peptide agonist according to any preceding claim, wherein Xaa ₁₉ is Abu. 8. A VPAC2 receptor peptide agonist according to any one of the preceding claims, wherein Xaa ₂₃ is Aib. 9. A VPAC2 receptor peptide agonist according to any preceding claim, wherein Xaa ₂₅ is Aib. 10. A VPAC2 receptor peptide agonist according to any preceding claim, wherein _Xaa29 , _Xaa30 , _Xaa31 and _Xaa32 are absent. 11. A VPAC2 receptor peptide agonist according to any one of the preceding claims, wherein said agonist is polyethylene glycol added. 12. A VPAC2 receptor peptide agonist according to any preceding claim, wherein said agonist is cyclic. 13. The VPAC2 receptor peptide agonist according to any one of the preceding claims, further comprising an N-terminal modification at the N-terminus of the peptide agonist, wherein said N-terminal modification is selected from: (a) adding D-histidine, isoleucine, methionine or norleucine; (b) adding a peptide containing the sequence Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg (SEQ ID NO: 6), wherein Arg is linked to the N-terminus of the peptide agonist; (c) Adding C ₁ -C ₁₆ alkyl optionally with one or more substituents independently selected from aryl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ elect to replace (d) Adding -C(O)R ¹ , wherein R ¹ is independently selected from aryl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen, -SH and -CF ₃ or multiple substituents optionally substituted C ₁ -C ₁₆ alkyl; independently selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ - Aryl optionally substituted by one or more substituents of C ₆ alkoxy, -NH ₂ , -OH, halogen, and -CF ₃ ; independently selected from C ₁ -C ₆ alkyl, C ₂ -C One or more substituents of ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ optionally substituted aryl C ₁ - C ₄ alkyl; -NR ² R ³ , wherein R ² and R ³ are independently hydrogen, C ₁ -C ₆ alkyl, aryl or arylC ₁ -C ₄ alkyl; -OR ⁴ , wherein R ⁴ is _{C 1} -C ₁₆ alkyl optionally substituted with one or more substituents independently selected from aryl, C 1 -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ , independently selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, -NH ₂ , -OH, halogen and -CF ₃ Optionally substituted aryl with one or more substituents independently selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkane One or more substituents of oxy, -NH ₂ , -OH, halogen and -CF ₃ optionally substituted aryl C ₁ -C ₄ alkyl; or 5-pyrrolidin-2-one; (e) adding -SO ₂ R ⁵ , wherein R ⁵ is aryl, aryl C ₁ -C ₄ alkyl or C ₁ -C ₁₆ alkyl; (f) forming a succinimide group optionally substituted with C ₁ -C ₆ alkyl or -SR ⁶ , wherein R ⁶ is hydrogen or C ₁ -C ₆ alkyl; (g) adding methionine sulfoxide; (h) adding biotinyl-6-aminocaproic acid (6-aminocarproicacid); and (i) Add -C(=NH)-NH ₂ . 14. The VPAC2 receptor peptide agonist according to claim 13, wherein the N-terminal modification is the addition of a group selected from the group consisting of acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine , Methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6 -aminocaproic acid (6-aminocaproic acid) and -C(=NH) _-NH2 . 15. The VPAC2 receptor peptide agonist according to claim 14, wherein said N-terminal modification is the addition of an acetyl group or a hexanoyl group. 16. The VPAC2 receptor peptide agonist according to claim 1, comprising the following amino acid sequence: Agonist# sequence P603 C6-HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQAibIOrnOrnGGPSSGAPPPK(E-C16)-NH ₂ 17. A pharmaceutical composition comprising a VPAC2 receptor peptide agonist according to any one of claims 1 to 16 together with one or more pharmaceutically acceptable diluents, carriers and excipients. 18. A VPAC2 receptor peptide agonist according to any one of claims 1 to 16 for use as a medicament. 19. A VPAC2 receptor peptide agonist according to any one of claims 1 to 16 for use in the treatment of non-insulin dependent diabetes or insulin dependent diabetes or for the suppression of food intake. 20. Use of a VPAC2 receptor peptide agonist according to any one of claims 1 to 16 for the manufacture of a medicament for the treatment of non-insulin-dependent diabetes or insulin-dependent diabetes or for the suppression of food intake . 21. A method of treating non-insulin-dependent diabetes or insulin-dependent diabetes or suppressing food intake in a patient in need thereof, said method comprising administering an effective amount of a VPAC2 receptor peptide according to any one of claims 1 to 16 agonist. 22. A pharmaceutical composition containing a VPAC2 receptor peptide agonist according to any one of claims 1 to 16 for the treatment of non-insulin-dependent diabetes or insulin-dependent diabetes or for the suppression of food intake . 23. A VPAC2 receptor peptide agonist substantially as hereinbefore described with reference to the Examples.